Department of Neuroscience, Biomedicine and Movement Sciences, University of Verona, Verona, Italy.
Department of Fundamental Neurosciences (DFN), University of Lausanne, Lausanne, Switzerland.
Brain Struct Funct. 2017 Nov;222(8):3847-3859. doi: 10.1007/s00429-017-1466-3. Epub 2017 Jul 1.
Orexin (OX)/hypocretin-containing neurons are main regulators of wakefulness stability, arousal, and energy homeostasis. Their activity varies in relation to the animal's behavioral state. We here tested whether such variation is subserved by synaptic plasticity phenomena in basal conditions. Mice were sacrificed during day or night, at times when sleep or wake, respectively, predominates, as assessed by electroencephalography in matched mice. Triple immunofluorescence was used to visualize OX-A perikarya and varicosities containing the vesicular glutamate transporter (VGluT)2 or the vesicular GABA transporter (VGAT) combined with synaptophysin (Syn) as a presynaptic marker. Appositions on OX-A somata were quantitatively analyzed in pairs of sections in epifluorescence and confocal microscopy. The combined total number of glutamatergic (Syn/VGluT2) and GABAergic (Syn/VGAT) varicosities apposed to OX-A somata was similar during day and night. However, glutamatergic varicosities were significantly more numerous at night, whereas GABAergic varicosities prevailed in the day. Triple immunofluorescence in confocal microscopy was employed to visualize synapse scaffold proteins as postsynaptic markers and confirmed the nighttime prevalence of VGluT2 together with postsynaptic density protein 95 excitatory contacts, and daytime prevalence of VGAT together with gephyrin inhibitory contacts, while also showing that they formed synapses on OX-A cell bodies. The findings reveal a daily reorganization of axosomatic synapses in orexinergic neurons, with a switch from a prevalence of excitatory innervation at a time corresponding to wakefulness to a prevalence of inhibitory innervations in the antiphase, at a time corresponding to sleep. This reorganization could represent a key mechanism of plasticity of the orexinergic network in basal conditions.
食欲素（OX）/下丘脑分泌素神经元是调节觉醒稳定性、觉醒和能量平衡的主要调节因子。它们的活动与动物的行为状态有关。我们在这里测试这种变化是否是在基础条件下通过突触可塑性现象来实现的。在匹配的小鼠中，通过脑电图评估睡眠或觉醒为主的时间，在白天或夜间处死小鼠。使用三重免疫荧光来可视化 OX-A 神经元体和含有囊泡谷氨酸转运体（VGluT）2 或囊泡 GABA 转运体（VGAT）的囊泡，并与突触小体蛋白（Syn）结合作为突触前标记。在荧光和共聚焦显微镜的成对切片中定量分析 OX-A 体上的突触前接触。白天和夜间的 OX-A 体上的谷氨酸能（Syn/VGluT2）和 GABA 能（Syn/VGAT）囊泡的总数相似。然而，夜间谷氨酸能囊泡的数量明显更多，而白天 GABA 能囊泡占优势。共聚焦显微镜的三重免疫荧光用于可视化突触支架蛋白作为突触后标记，并证实了 VGluT2 与突触后密度蛋白 95 兴奋性接触的夜间优势，以及 VGAT 与 Gephyrin 抑制性接触的日间优势，同时还表明它们在 OX-A 神经元体上形成了突触。这些发现揭示了食欲素能神经元中轴突-体突触的日常重组，从与觉醒相对应的时间的兴奋性支配优势转变为与睡眠相对应的时间的抑制性支配优势。这种重组可能代表基础条件下食欲素能网络可塑性的关键机制。
