Evans Jenafer, Sumners Colin, Moore Jennifer, Huentelman Matthew J, Deng Jie, Gelband Craig H, Shaw Gerry
Department of Physiology, McKnight Brain Institute, University of Florida College of Medicine, Gainesville, FL 32610, USA.
J Neurophysiol. 2002 Feb;87(2):1076-85. doi: 10.1152/jn.00088.2001.
Embryonic or neonatal rat neurons retain plasticity and are readily grown in tissue culture, but neurons of the adult brain were thought to be terminally differentiated and therefore difficult to culture. Recent studies, however, suggest that it may be possible to culture differentiated neurons from the hippocampus of adult rats. We modified these procedures to grow differentiated neurons from adult rat hypothalamus and brain stem. At day 7 in tissue culture and beyond, the predominant cell types in hypothalamic and brain stem cultures had a stellate morphology and could be subdivided into two distinct groups, one of which stained with antibodies to the immature neuron marker alpha-internexin, while the other stained with the astrocyte marker GFAP. The alpha-internexin positive cells were mitotic and grew to form a characteristic two-dimensional cellular network. These alpha-internexin positive cells coimmunostained for the neuronal markers MAP2, type III beta-tubulin, and tau, and also bound tetanus toxin, but were negative for the oligodendrocyte marker GalC and also for the neurofilament triplet proteins NF-L, NF-M, and NF-H, markers of more mature neurons. Patch-clamp analysis of these alpha-internexin positive cells revealed small Ca(2+) currents with a peak current of -0.5 +/- 0.1 pA/pF at a membrane potential of -20 mV (n = 5) and half-maximal activation at -30 mV (n = 5). Na(+) currents with a peak current density of -154.5 +/- 49.8 pA/pF at a membrane potential of -15 mV (n = 5) were also present. We also show that these cells can be frozen and regrown in tissue culture and that they can be efficiently infected by viral vectors. These cells therefore have the immunological and electrophysiological properties of immature mitotic neurons and should be useful in a variety of future studies of neuronal differentiation and function.
胚胎期或新生大鼠的神经元具有可塑性，易于在组织培养中生长，但成体大脑的神经元被认为是终末分化的，因此难以培养。然而，最近的研究表明，有可能培养成年大鼠海马体中已分化的神经元。我们改进了这些方法，以培养成年大鼠下丘脑和脑干中已分化的神经元。在组织培养第7天及以后，下丘脑和脑干培养物中的主要细胞类型具有星状形态，可分为两个不同的组，其中一组用未成熟神经元标记物α-中间丝蛋白的抗体染色，而另一组用星形胶质细胞标记物GFAP染色。α-中间丝蛋白阳性细胞可进行有丝分裂，并生长形成特征性的二维细胞网络。这些α-中间丝蛋白阳性细胞与神经元标记物MAP2、III型β-微管蛋白和tau共免疫染色，也结合破伤风毒素，但对少突胶质细胞标记物GalC以及更成熟神经元的标记物神经丝三联蛋白NF-L、NF-M和NF-H呈阴性。对这些α-中间丝蛋白阳性细胞进行膜片钳分析发现，在膜电位为-20 mV（n = 5）时，有小的Ca(2+)电流，峰值电流为-0.5 +/- 0.1 pA/pF，并在-30 mV时达到半数最大激活（n = 5）。在膜电位为-15 mV（n = 5）时，也存在峰值电流密度为-154.5 +/- 49.8 pA/pF的Na(+)电流。我们还表明，这些细胞可以冷冻并在组织培养中重新生长，并且它们可以被病毒载体有效感染。因此，这些细胞具有未成熟有丝分裂神经元的免疫学和电生理学特性，在未来各种神经元分化和功能研究中应该会很有用。
