Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA269

Poster Communications

Visualization of morphological changes of PSD95 substructures in the living mouse brain by STED microscopy

H. Steffens1,2,3, W. Wegner1,2,3, A. Mott1,3, K. I. Willig1,2,3

1. University Medical Center Göttingen, Optical Nanoscopy in Neuroscience, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Göttingen, Germany. 2. University of Göttingen, Collaborative Research Center 889, Göttingen, Germany. 3. Max Planck Institute of Experimental Medicine, Göttingen, Germany.

Far-field light microscopy is a powerful technique for imaging structures inside living cells, tissue or living animals. However, fine details or substructures of the cell cannot be visualized because of diffraction limited resolution (~200-350 nm). From all novel light microscopy or super-resolution microscopy techniques available presently, STED (STimulated Emission Depletion) nanoscopy stands out for its imaging capabilities in tissue. Here, we used STED nanoscopy to determine short term changes in size and shape of PSD95 in the anesthetized mouse visual cortex. These morphological changes should relate to variations of synaptic strength, and the formation or elimination of synapses, respectively (El-Husseini et al. 2000; Meyer et al. 2014). Adult knock-in mice expressing eGFP fused to the endogenous PSD95 protein were imaged at time points from 1 min to 6 h. All experiments were performed according to ethical rules of the European Union and the guidelines of the national law (Tierschutzgesetz der Bundesrepublik Deutschland, TierSchG) regarding animal protection procedures. General anesthesia was initiated by i.p. injection of 60-80 mg pentobarbital sodium (in 0.9% NaCl) per kg body weight. Once the mouse was anesthetized, the right jugular vein was cannulated, and anesthesia was continued with 75 mg kg−1 h−1 methohexital sodium (Brevimytal®, HIKMA, Gräfelfing, Germany) i.v. throughout the experiment. A tracheotomy was performed to intubate the mouse. The mouse was paralyzed with pancuronium bromide (6 mg kg−1 h−1) and connected to artificial ventilation at 100-120 breaths per minute and a breath volume of 100-140 μl to avoid movements by active respiration. Body temperature was rectally measured and controlled by a heat plate. Super-resolved large assemblies of PSD95 show different sub-structures; most large assemblies were ring-like, some horseshoe or figure-8 shaped, and shapes were continuous or made up of nanoclusters. The sub-structure appeared stable during the shorter (minute) time points, but after 1 h, more than 50% of the large assemblies showed a change in sub-structure. The morphing of the PSD95 assemblies did not occur continuously; some assemblies where stable over 3 h but then changed after another 3 h, others changed and then regained their original shape. Within our imaging time course of 6 h 90% of the large PSD95 assemblies underwent a morphological change. Overall, these data showed a sub-morphology of large PSD95 assemblies which undergo changes within the 6 hours of observation in the anaesthetized mouse. At this time we can only speculate on the impact of these changes on the synaptic machinery and therefore did not quantify it further. To further analyze the physiological background of changes it would be necessary to perform experiments with defined stimulation or deprivation as possible triggers for these changes.

Where applicable, experiments conform with Society ethical requirements