Cowan R L, Sesack S R, Van Bockstaele E J, Branchereau P, Chain J, Pickel V M
Department of Neurology and Neuroscience, Cornell University Medical College, New York, New York 10021.
Synapse. 1994 Jun;17(2):101-14. doi: 10.1002/syn.890170206.
Ultrastructural immunocytochemical identification of transmitters in afferent terminals and targets of individual physiologically characterized neurons is essential for understanding the complex circuitry within the mammalian neocortex. For this type of analysis, we examined the utility of combining in vivo intracellular recording and biocytin injections with silver intensified 1 nm immunogold labeling of GABA and the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH). These transmitters are found to local neurons and afferents known to prominently modulate the activity of pyramidal neurons in the neocortex. Individual neurons were physiologically characterized and filled with biocytin in the frontal cortex of anesthetized rats. The brains were then preserved by vascular perfusion with aldehydes. Single vibratome sections through the recording site were reacted (1) for immunoperoxidase detection of biocytin and (2) for immunogold labeling of GABA or TH. Dually labeled sections were processed for light microscopy or embedded in plastic for electron microscopy. The dense peroxidase product for biocytin was detected in pyramidal neurons. These were located in superficial as well as deep cortical laminae, and were readily distinguished from immunogold silver labeling. GABA labeled terminals formed symmetric synapses with larger biocytin filled dendrites, whereas the TH labeled terminals contacted distal dendrites and spines. Peroxidase labeling for biocytin also was seen in a few axon terminals forming synapses with unlabeled and with GABA immunoreactive dendrites. These results suggest that single pyramidal neurons of the rat frontal cortex receive dual input from both GABA and catecholamine terminals. Additionally, this study demonstrates the usefulness of silver enhancement of 1 nm colloidal gold prior to plastic embedding for electron microscopic detection of neurotransmitters within afferents and targets of neurons physiologically characterized in vivo.
超微结构免疫细胞化学鉴定传入终末和单个生理特征明确的神经元靶标的递质，对于理解哺乳动物新皮质内复杂的神经回路至关重要。对于此类分析，我们研究了将体内细胞内记录和生物胞素注射与GABA和儿茶酚胺合成酶酪氨酸羟化酶（TH）的银增强1纳米免疫金标记相结合的实用性。这些递质存在于已知对新皮质锥体神经元活动有显著调节作用的局部神经元和传入纤维中。在麻醉大鼠的额叶皮质对单个神经元进行生理特征鉴定并注入生物胞素。然后通过醛类血管灌注保存大脑。通过记录部位的单张振动切片进行如下反应：（1）用于生物胞素的免疫过氧化物酶检测；（2）用于GABA或TH的免疫金标记。对双重标记的切片进行光学显微镜处理或包埋在塑料中用于电子显微镜观察。在锥体神经元中检测到生物胞素的密集过氧化物酶产物。这些锥体神经元位于皮质浅层和深层，很容易与免疫金银标记区分开来。GABA标记的终末与较大的生物胞素填充树突形成对称突触，而TH标记的终末与远端树突和棘突接触。在一些与未标记和GABA免疫反应性树突形成突触的轴突终末中也可见生物胞素的过氧化物酶标记。这些结果表明，大鼠额叶皮质的单个锥体神经元接受来自GABA和儿茶酚胺终末的双重输入。此外，本研究证明了在塑料包埋前对1纳米胶体金进行银增强，对于电子显微镜检测体内生理特征明确的神经元传入纤维和靶标中的神经递质是有用的。
