Keifer J, Vyas D, Houk J C, Berrebi A S, Mugnaini E
Department of Physiology, Northwestern University Medical School, Chicago, Illinois 60611.
Synapse. 1992 Jul;11(3):197-213. doi: 10.1002/syn.890110305.
Immunocytochemical and electrophysiological evidence supporting the presence of GABAergic interneurons in the turtle red nucleus is presented. Injections of HRP into the spinal cord produced labeling of large neurons in the contralateral red nucleus. The peroxidase-antiperoxidase (PAP) method revealed smaller cells immunoreactive to an antibody against glutamate decarboxylase (GAD), the synthetic enzyme for the inhibitory neurotransmitter GABA, that were interspersed among larger immunonegative neurons. Similar small neurons were densely immunostained by antibodies to GABA-glutaraldehyde conjugates obtained from different sources and applied according to pre-embedding and postembedding protocols. Rubrospinal neurons retrogradely labeled with HRP measured 16 and 27 microns in mean minor and major cell body diameters, while GABA-like immunopositive neurons situated within the red nucleus measured 7 and 13 microns. There was very little overlap in soma size between the two cell populations. Therefore, we suggest that the GAD- and GABA-positive neurons may be local inhibitory interneurons. This notion is further supported by observations of pre-embedding immunostaining for GAD and postembedding immunostaining for GABA showing that the turtle red nucleus is amply innervated by immunoreactive axon terminals. These puncta are closely apposed to cell bodies and dendrites of both immunonegative large neurons and immunopositive small neurons. Moreover, immunogold staining at the electron microscopic level demonstrated that GABA-like immunoreactive axon terminals with pleomorphic synaptic vesicles formed symmetric synapses with cell bodies and dendrites of the two types of red nucleus cells. These ultrastructural features are commonly assumed to indicate inhibitory synapses. A moderately labeled bouton with round vesicles and asymmetric synapses was also observed. In addition, the two types of red nucleus neurons received asymmetric axosomatic and axodendritic synapses with GABA-negative boutons provided with round vesicles, features usually associated with excitatory functions. To obtain electrophysiological evidence for inhibition, intracellular recordings from red nucleus neurons were conducted using an in vitro brainstem-cerebellum preparation from the turtle. Small, spontaneous IPSPs were recorded from 7 out of 14 red nucleus cells studied. These morphological and physiological results provide strong support for concluding that the turtle red nucleus, like its mammalian counterpart, contains GABAergic inhibitory interneurons. While we have not identified the main source of input to these interneurons, in view of the scarce development of the reptilian cerebral cortex, this input is unlikely to come from the motor cortex as it does in mammals.(ABSTRACT TRUNCATED AT 400 WORDS)
本文提供了免疫细胞化学和电生理学证据，支持龟的红核中存在γ-氨基丁酸（GABA）能中间神经元。将辣根过氧化物酶（HRP）注入脊髓，可使对侧红核中的大神经元产生标记。过氧化物酶-抗过氧化物酶（PAP）方法显示，对谷氨酸脱羧酶（GAD，抑制性神经递质GABA的合成酶）抗体呈免疫反应的较小细胞散布在较大的免疫阴性神经元之间。用从不同来源获得的GABA-戊二醛缀合物抗体，按照包埋前和包埋后方案进行处理，类似的小神经元被密集免疫染色。用HRP逆行标记的红核脊髓神经元，其平均小和大细胞体直径分别为16和27微米，而位于红核内的GABA样免疫阳性神经元直径为7和13微米。这两种细胞群体的体细胞大小几乎没有重叠。因此，我们认为GAD和GABA阳性神经元可能是局部抑制性中间神经元。对GAD的包埋前免疫染色和对GABA的包埋后免疫染色观察进一步支持了这一观点，表明龟的红核有丰富的免疫反应性轴突终末支配。这些小点紧密贴附于免疫阴性大神经元和免疫阳性小神经元的细胞体和树突。此外，电子显微镜水平的免疫金染色表明，具有多形性突触小泡的GABA样免疫反应性轴突终末与两种红核细胞的细胞体和树突形成对称突触。这些超微结构特征通常被认为表明是抑制性突触。还观察到一个带有圆形小泡和不对称突触的中等标记终扣。此外，两种红核神经元都接受了与带有圆形小泡的GABA阴性终扣形成的不对称轴体和轴树突触，这些特征通常与兴奋功能相关。为了获得抑制的电生理学证据，使用龟的离体脑干-小脑标本对红核神经元进行细胞内记录。在所研究的14个红核细胞中，有7个记录到了小的自发性抑制性突触后电位（IPSP）。这些形态学和生理学结果为得出龟的红核与哺乳动物的红核一样含有GABA能抑制性中间神经元的结论提供了有力支持。虽然我们尚未确定这些中间神经元的主要输入来源，但鉴于爬行动物大脑皮层发育稀少，这种输入不太可能像在哺乳动物中那样来自运动皮层。（摘要截断于400字）
