Müller M, Somjen G G
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Neurophysiol. 1999 Oct;82(4):1818-31. doi: 10.1152/jn.1999.82.4.1818.
In interfaced rat hippocampal slices spreading depression (SD) and hypoxia-induced SD-like depolarization are associated with increased light reflectance and decreased light transmittance, indicating increased light scattering. By contrast, mild hypotonicity or electrical stimulation decrease light scattering, which is usually taken to be caused by cell swelling. This difference has been attributed to experimental conditions, but in our laboratory moderate osmotic challenge and SD produced opposite intrinsic optical signals (IOSs) in the same slice under identical conditions. To decide whether the SD-induced IOS is related to cell swelling, we investigated the effects of Cl(-) transport inhibitors and Cl(-) withdrawal on both light reflectance and transmittance, as well as on changes in interstitial volume and tissue electrical resistance. In normal Cl(-), early during hypoxia, there was a slight decrease in light reflectance paired with increase in transmittance. At the onset of hypoxic SD, coincident with the onset of cell swelling (restriction of TMA(+) space), the IOS signals suddenly inverted, indicating sharply increased scattering. The SD-related IOSs started in a single spot and spread out over the entire CA1 region without invading CA3. Application of 2 mM furosemide decreased IOS intensity. When Cl(-) was substituted by methylsulfate or gluconate, the SD-related reflectance increase and transmittance decrease were suppressed and replaced by opposite signals, indicating scattering decrease. Yet Cl(-) withdrawal did not prevent cell swelling measured as shrinkage of TMA(+) space. The SD-related increase of tissue electrical resistance was reduced when bath Cl(-) was replaced by methylsulfate and almost eliminated when replaced by gluconate. The TMA(+) signal is judged to be a more reliable indicator of interstitial space than tissue resistance. Neither application of cyclosporin A nor raising Mg(2+) depressed the SD-related reflectance increase, suggesting that Cl(-) flux through mitochondrial "megachannels" may not be a major factor in its generation. Fluoroacetate poisoning of glial cells (5 mM) accelerated SD onset and enhanced the SD-induced reflectance increase threefold. This suggests, first, that glial cells normally moderate the SD process and, second, that neurons are the predominant generators of the light-scattering increase. We conclude that light scattering by cerebral tissue can be changed by at least two different physical processes. Cell swelling decreases light scattering, whereas a second process increases scattering. During hypoxic SD the scattering increase masks the swelling-induced scattering decrease, but the latter is revealed when Cl(-) is removed. The scattering increase is Cl(-) dependent, nevertheless it is apparently not related to cell volume changes. Its underlying mechanism is as yet not clear; possible factors are discussed.
在连接的大鼠海马切片中，扩散性抑制（SD）和缺氧诱导的类SD去极化与光反射率增加和透光率降低相关，表明光散射增加。相比之下，轻度低渗或电刺激会降低光散射，这通常被认为是由细胞肿胀引起的。这种差异归因于实验条件，但在我们实验室中，在相同条件下，中等渗透挑战和SD在同一切片中产生了相反的固有光学信号（IOS）。为了确定SD诱导的IOS是否与细胞肿胀有关，我们研究了Cl⁻转运抑制剂和Cl⁻去除对光反射率和透光率以及细胞间液体积和组织电阻变化的影响。在正常的[Cl⁻]ₒ中，缺氧早期，光反射率略有下降，同时透光率增加。在缺氧性SD开始时，与细胞肿胀开始（TMA⁺空间受限）同时发生，IOS信号突然反转，表明散射急剧增加。与SD相关的IOS从单个点开始，在整个CA1区域扩散，而不侵入CA3。应用2 mM速尿可降低IOS强度。当[Cl⁻]ₒ被甲硫酸盐或葡萄糖酸盐替代时，与SD相关的反射率增加和透光率降低受到抑制，并被相反的信号取代，表明散射减少。然而，Cl⁻去除并不能阻止以TMA⁺空间收缩衡量的细胞肿胀。当浴液中的Cl⁻被甲硫酸盐替代时，与SD相关的组织电阻增加减少，当被葡萄糖酸盐替代时几乎消除。TMA⁺信号被认为比组织电阻更可靠地指示细胞间空间。应用环孢素A或提高[Mg²⁺]ₒ均未抑制与SD相关的反射率增加，这表明通过线粒体“大通道”的Cl⁻通量可能不是其产生的主要因素。用氟乙酸盐毒害神经胶质细胞（5 mM）加速了SD的开始，并使SD诱导的反射率增加了三倍。这表明，首先，神经胶质细胞通常会调节SD过程，其次，神经元是光散射增加的主要产生者。我们得出结论，脑组织的光散射可以通过至少两种不同的物理过程发生变化。细胞肿胀会降低光散射，而另一个过程会增加散射。在缺氧性SD期间，散射增加掩盖了肿胀诱导的散射减少，但当Cl⁻被去除时，后者会显现出来。散射增加依赖于Cl⁻，但其显然与细胞体积变化无关。其潜在机制尚不清楚；讨论了可能的因素。
