Horvath, O. | bioRxiv Science

bioRxiv2023

A sample preparation procedure enables acquisition of 2-channel super-resolution 3D STED image of an entire oocyte

Super-resolution (SR) microscopy is a cutting-edge method that can provide detailed structural information with high resolution. However, the thickness of the specimen has been a major limitation for SR methods, and larger structures have posed a challenge. To overcome this, the key step is to optimize sample preparation to ensure optical homogeneity and clarity, which can enhance the capabilities of SR methods for the acquisition of thicker structures. Oocytes are the largest cells in the mammalian body and are crucial objects in reproductive biology. They are especially useful for studying membrane proteins. However, oocytes are extremely fragile and sensitive to mechanical manipulation and osmotic shocks, making sample preparation a critical and challenging step. We present an innovative, simple, and sensitive approach to oocyte sample preparation for 3D STED acquisition. This involves alcohol dehydration and mounting into a high refractive index medium. This extended preparation procedure allowed us to successfully obtain a unique 2-channel 3D STED super-resolution image of an entire mouse oocyte. By optimizing sample preparation, we can overcome the limitations of SR methods and obtain high-resolution images of larger structures, such as oocytes, Knowledge of which are important for understanding fundamental biological processes. RESEARCH HIGHLIGHTSO_LIThis study aimed to develop a successful sample preparation protocol for imaging mouse oocytes using 3D STED super-resolution microscopy. C_LIO_LIThe results showed that the oocyte sample was optically homogenous, enhancing the capability of the 3D STED method to capture high-resolution images throughout the full depth of the sample, resulting in highly similar SR images. C_LIO_LIThe 3D STED point spread function (PSF) pattern of the depletion laser was successfully secured throughout the entire volume of the sample, including the top and bottom. C_LIO_LIThis study presents the first-ever full volume 3D image of the mouse oocyte, which was acquired using a 2-channel 3D STED method. C_LI GRAPHICAL ABSTRACTIntroducing an extended sample preparation procedure resulted in an outstanding optical quality sample environment pronounced by high refractive index, high transparency, and minimal spherical aberration. This procedure allowed 3D STED within the entire oocyte. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

cell biology↗

bioRxiv2020

Somatostatin neurons of the bed nucleus of stria terminalis enhance associative fear memory consolidation in mice

Excessive fear learning and extinction-resistant fear memories are core symptoms of anxiety and trauma-related disorders. Despite significant evidence from clinical studies reporting hyperactivity of the bed nucleus of stria terminalis (BNST) under these conditions, the role of BNST in fear learning and expression is still not clarified. Here, we tested how BNST modulates fear learning in mice using a chemogenetic approach. Activation of GABAergic neurons of BNST during fear acquisition, more specifically the consolidation phase, resulted in enhanced cued fear recall. Importantly, BNST activation had no acute impact on fear expression during conditioning or recalls, but it enhanced cued fear recall subsequently, potentially via altered activity of downstream regions as indicated by c-Fos. Enhanced fear memory consolidation could be replicated by selectively activating somatostatin neurons (but not corticotropin releasing factor neurons), suggesting significant modulation of fear memory strength by specific circuits of BNST.

neuroscience↗