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.
“Direct” stochastic optical reconstruction microscopy (dSTORM) is a super-resolution microscopy approach for imaging biological samples in which fluorescent proteins are genetically expressed in the cell of interest. The method is based on repeated imaging of individual fluorescent molecules within the sample, using stochastic illumination and image acquisition at a rate of a few frames per second to minimize imaging artifacts. This technique requires a number of additional technical considerations, including an appropriate microscope setup with suitable excitation and emission wavelengths, imaging buffer, and image processing software, which take several hours to perform. The technique is particularly well suited for imaging biological samples in cells and tissues, as these samples contain a high density of molecules. The resolution achieved is largely determined by the number of fluorescent molecules imaged per pixel, the localization precision (i.e. the accuracy of locating a specific molecule in the sample) and the number of frames per second imaged. 827ec27edc