Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA.
J Neurosci Methods. 2012 Mar 30;205(1):130-8. doi: 10.1016/j.jneumeth.2011.12.017. Epub 2012 Jan 2.
We have recently reported that in the learned helplessness model of depression, the less hippocampal spine synapses rats have, the more helpless they become. It remains unclear, however, whether the observed synaptic changes are associated with the loss of CA1 pyramidal cells. Cell bodies in the CA1 pyramidal layer are very densely packed, making cell counting difficult in this hippocampal subregion. To address this issue, we developed a new approach that (1) yields excellent preservation of the three-dimensional tissue structure; (2) utilizes osmium tetroxide to unambiguously label nucleoli; and (3) facilitates and accelerates unbiased, reliable counting of densely packed cell bodies. Our method provides an improved tool for studies aiming to evaluate hippocampal atrophy and cell loss, the most characteristic features in many neurodegenerative diseases, such as Alzheimer's disease, temporal lobe epilepsy and ischemia, as well as in several psychiatric disorders. Using this new method, we demonstrated no significant changes in the number of CA1 pyramidal cells in the rat learned helplessness paradigm. In addition, volumes of the CA1 pyramidal cell layer and the entire CA1 subfield remained unchanged among treatment groups. We conclude that previously observed synaptic alterations in helpless rats are not associated with CA1 pyramidal cell loss. This finding suggests that behavioral outcome in the learned helplessness paradigm is related to plastic events at the synaptic level, rather than at the level of principal cells.
我们最近报道，在抑郁的习得性无助模型中，海马体棘突触较少的大鼠变得更加无助。然而，目前尚不清楚观察到的突触变化是否与 CA1 锥体神经元的丢失有关。CA1 锥体层的细胞体非常密集，使得在这个海马亚区进行细胞计数变得困难。为了解决这个问题，我们开发了一种新方法，（1）可以极好地保持三维组织结构；（2）利用四氧化锇明确标记核仁；（3）便于加快并促进对密集排列的细胞体进行无偏倚、可靠的计数。我们的方法为评估海马体萎缩和细胞丢失的研究提供了一种改进的工具，这些是许多神经退行性疾病（如阿尔茨海默病、颞叶癫痫和缺血）以及几种精神疾病的最典型特征。使用这种新方法，我们在习得性无助大鼠模型中没有发现 CA1 锥体神经元数量的显著变化。此外，CA1 锥体细胞层和整个 CA1 亚区的体积在各组之间没有变化。我们得出结论，先前在无助大鼠中观察到的突触改变与 CA1 锥体神经元的丢失无关。这一发现表明，习得性无助模型中的行为结果与突触水平上的可塑性事件有关，而不是与主细胞有关。
