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Ultradian Secretion of Growth Hormone in Mice: Linking Physiology With Changes in Synapse Parameters Using Super-Resolution Microscopy.
Frontiers in Neural Circuits ( IF 3.5 ) Pub Date : 2020-04-14 , DOI: 10.3389/fncir.2020.00021
Klaudia Bednarz 1, 2 , Walaa Alshafie 1, 3 , Sarah Aufmkolk 4 , Théotime Desserteaux 1 , Pratap Singh Markam 3, 5 , Kai-Florian Storch 5, 6 , Thomas Stroh 1, 2
Affiliation  

Neuroendocrine circuits are orchestrated by the pituitary gland in response to hypothalamic hormone-releasing and inhibiting factors to generate an ultradian and/or circadian rhythm of hormone secretion. However, mechanisms that govern this rhythmicity are not fully understood. It has been shown that synaptic transmission in the rodent hypothalamus undergoes cyclical changes in parallel with rhythmic hormone secretion and a growing body of evidence suggests that rapid rewiring of hypothalamic neurons may be the source of these changes. For decades, structural synaptic studies have been utilizing electron microscopy, which provides the resolution suitable for visualizing synapses. However, the small field of view, limited specificity and manual analysis susceptible to bias fuel the search for a more quantitative approach. Here, we apply the fluorescence super-resolution microscopy approach direct Stochastic Optical Reconstruction Microscopy (dSTORM) to quantify and structurally characterize excitatory and inhibitory synapses that contact growth hormone-releasing-hormone (GHRH) neurons during peak and trough values of growth hormone (GH) concentration in mice. This approach relies on a three-color immunofluorescence staining of GHRH and pre- and post-synaptic markers, and a quantitative analysis with a Density-Based Spatial Clustering of Applications with Noise (DBSCAN) algorithm. With this method we confirm our previous findings, using electron microscopy, of increased excitatory synaptic input to GHRH neurons during peak levels of GH. Additionally, we find a shift in synapse numbers during low GH levels, where more inhibitory synaptic inputs are detected. Lastly, we utilize dSTORM to study novel aspects of synaptic structure. We show that more excitatory (but not inhibitory) pre-synaptic clusters associate with excitatory post-synaptic clusters during peaks of GH secretion and that the numbers of post-synaptic clusters increase during high hormone levels. The results presented here provide an opportunity to highlight dSTORM as a valuable quantitative approach to study synaptic structure in the neuroendocrine circuit. Importantly, our analysis of GH circuitry sheds light on the potential mechanism that drives ultradian changes in synaptic transmission and possibly aids in GH pulse generation in mice.



中文翻译:

小鼠生长激素的超分子分泌:使用超分辨显微镜将生理学与突触参数的变化联系起来。

垂体对下丘脑激素的释放和抑制因子作出响应,由垂体协调神经内分泌回路,以产生激素分泌的超昼夜节律和/或昼夜节律。但是,尚不完全了解控制这种节奏的机制。已经显示,啮齿类动物下丘脑中的突触传递与节律性激素分泌同时经历周期性变化,越来越多的证据表明,下丘脑神经元的快速重新布线可能是这些变化的来源。几十年来,结构突触研究一直利用电子显微镜,其提供了适合于可视化突触的分辨率。然而,视野狭小,特异性有限以及易于偏见的人工分析推动了寻找更定量方法的努力。这里,直接 随机光学重建显微镜d(STORM)来量化和结构表征在小鼠中生长激素(GH)浓度的峰值和谷值期间接触生长激素释放激素(GHRH)神经元的兴奋性和抑制性突触。这种方法依赖于GHRH和突触前和突触后标记的三色免疫荧光染色,以及基于密度的基于噪声的应用程序空间聚类(DBSCAN)算法的定量分析。通过这种方法,我们使用电子显微镜证实了我们先前的发现,即在GH峰值水平期间,向GHRH神经元的兴奋性突触输入增加。此外,我们发现在低GH水平期间,突触数量发生了变化,其中检测到更多抑制性突触输入。最后,我们利用dSTORM研究突触结构的新方面。我们显示,在GH分泌高峰期间,更多的兴奋性(但不是抑制性)突触前丛集与兴奋性突触后丛集相关,并且在高激素水平期间,突触后丛集的数量增加。此处呈现的结果提供了一个突出显示的机会dSTORM是研究神经内分泌回路中突触结构的一种有价值的定量方法。重要的是,我们对GH电路的分析揭示了驱动突触传递中超原性变化的潜在机制,并可能有助于小鼠中GH脉冲的产生。

更新日期:2020-04-14
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