Nanoscopic Imaging of Neuroendocrine Regulatory Processes Using Super-Resolution Microscopy

Nanoscopic Imaging of Neuroendocrine Regulatory Processes in the Hypothalamo-Pituitary Axis Using dSTORM

Single molecule localization microscopy (SMLM) using direct Stochastic Optical Reconstruction Microscopy (dSTORM) enables the imaging of regulatory processes in the brain at the near-molecular level, which cannot be resolved using traditional fluorescence microscopy techniques. However, imaging brain samples is challenging due to the optical density of the samples. Nevertheless, suitable tissue clearing, biplane imaging to capture 3D data, as well as computational and statistical methods to differentiate between signal and noise can overcome these difficulties. 

Here, we used biplane SMLM and dSTORM in combination with highly sensitive determination of plasma hormone levels to investigate receptor trafficking and synaptic remodeling within the hypothalamo-pituitary endocrine circuit in endocrine pituitary cells and brain tissue, respectively. In corticotropic pituitary cells, we identified a novel syntaxin-6 positive compartment that is distinct from the trans-Golgi-network (TGN) by dSTORM. This compartment facilitates the regulated recycling of the somatostatin receptor subtype 2 (SSTR2) to the cell surface following Corticotropin-Releasing Hormone stimulation, using nearest-neighbor analysis to determine the size of this compartment. In the hypothalamus, we used dSTORM to image synaptic connectivity and its remodeling in sections through the arcuate nucleus of the mouse hypothalamus in parallel with the pulsatile rhythm of Growth Hormone (GH) secretion, employing biplane imaging and the DBSCAN clustering algorithm. We observed that during peak GH secretion, there were more excitatory synapses at the somata of GH-releasing hormone (GHRH) neurons.

Conversely, during periods of low GH levels, inhibitory synaptic input increased on GHRH cells. The results presented here highlight biplane imaging and dSTORM as a valuable quantitative approach to study receptor trafficking and synaptic structure in neuroendocrine circuits. Using appropriate sample preparation, SMLM imaging techniques, and careful statistical analysis we achieved a nanometer-scale precision in identifying a novel compartment in the pituitary cells as well as imaging and quantifying synaptic remodeling under the influence of hormonal secretion cycles in mouse brain.

Date: Wednesday, November 27
Time: 8AM PST | 11AM EST | 5PM CET
Location: Online
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