Presented by: Randy Stout, Ph.D., Associate Professor, Department of Biomedical Sciences, New York Institute of Technology, with a brief introduction to ZEISS Dynamics Profiler by Samantha Fore, Product Marketing Manager LSM Life Sciences, Carl Zeiss Microscopy

Connexin proteins form gap junctions that directly connect astrocytes and oligodendrocytes into a cellular network. Neurons also connect via a different set of connexins but do not connect to astrocytes except in some rare developmental stages and brain regions. A major astrocyte connexin 43 (Cx43, GJA1) is highly expressed in the brain and most other organs; Cx43 mutations in humans produce skeletal malformations, white matter abnormalities, and highly variable cognitive deficits. Loss of oligodendrocyte Cx47 in humans leads to the variable but severe demyelinating brain disorder called Pelizaeus-Merzbacher Like Disease. Changes in gap junction expression and subcellular localization have been reported in humans and mouse models of brain diseases including major depression, autism spectrum disorder, traumatic brain injury, multiple sclerosis, Alzheimer’s disease, and many others. Clearly astrocyte gap junctions are important in human brain health and should be better understood.
Along with other labs, we have used high-resolution light microscopy to study the dynamics of gap junction supramolecular complexes and their protein binding partners. We used super-resolution confocal microscopy and Fluorescence Recovery After Photobleach (FRAP), respectively, to study the structure and dynamics of gap junctions. These imaging tools allowed new insights into the molecular mechanisms underlying gap junction nexus structure and dynamics. Most of the experiments to test these new ideas were performed with gap junctions of exaggerated scale and in non-neural cell lines because measuring the movement of Cx43 channels and their binding partners in the relevant small cellular spaces/scales exhibited by astrocytes in brain tissue (less than five micrometers in diameter) is challenging or impossible with FRAP or other techniques used to quantify protein mobility. Therefore, we are now using Zeiss Microscopy’s exciting new extended fluorescence correlation spectroscopy tool called Dynamics Profiler to quantify the number and mobility of proteins that interact with gap junctions in several distinct cellular compartments of micrometer scales. We will show unpublished data on the directionality of Cx43 interacting proteins changes at boundaries in the gap junction nexus supramolecular complex. More accurate understanding of protein dynamics at the molecular scale will be essential to building and interpreting computational models of astrocyte contributions to brain activity in health and disease. 

Presented by: Mehwish Anwer, Ph.D., Postdoctoral Fellow, Department of Pathology and Laboratory Medicine, University of British Columbia, Canada

Traumatic brain injury (TBI) induces a myriad of pathological processes resulting in brain-wide damage to cellular activity, axonal connectivity, and vasculature integrity. Despite the fundamental importance of understanding impaired brain activity exhibited in post-traumatic epilepsy and other neurological impairments associated with TBI, knowledge of how brain injury affects neuronal activity remains remarkably incomplete. We developed a whole-brain imaging and analysis approach to identify alterations in neuronal activity after TBI as a complementary method to conventional two-dimensional histological approaches. We established an easy-to-follow experimental pipeline to quantify changes in the whole mouse brain using tissue clearing, light sheet microscopy and an optimised open-access atlas registration workflow.
Using the CHIMERA (Closed-Head Impact Model of Engineered Rotational Acceleration) TBI model, TRAP2 mice were subjected to repeated mild TBI or sham treatment followed by tamoxifen injection to lock c-Fos activity after TBI. Brains were SHIELD fixed and passively cleared for imaging of c-Fos+ cells throughout the rostro-caudal axis of the brain using a light sheet microscope equipped with a specialized whole-brain imaging chamber. Volumetric images were stitched and 3D rendered using Arivis Vision4D image analysis software. For quantitative analysis, 2D image stacks were exported to segment c-Fos+ cells and register them to the Allen Mouse Brain Atlas using the BrainQuant3D python package. As a result, c-Fos+ cell counts were estimated throughout the brain and heatmaps were generated. A brain-wide reduction in c-Fos cell density was identified in the TBI group compared to sham controls, indicative of TBI-induced changes in whole brain neuronal activity. Further studies using multi-dimensional imaging of axonal and vascular damage coupled with 3D analysis tools will deepen our understanding of post-TBI brain-wide dynamics.

Presented by: Jarret AP Weinrich, Madison E Jewell, and Allan I Basbaum, Department of Anatomy, University of California San Francisco

General anesthetics are potent regulators of pain, but how they affect specific pain features, such as aversion, is not well understood. In this study, we investigated the effect of isoflurane, a general anesthetic, on the anterior cingulate cortex (ACC), a brain region implicated in generating pain's negative affect. Using in vivo imaging of neuronal calcium dynamics in mice, we found that spontaneous ACC activity persists at concentrations of isoflurane that induce loss of behavioral responsiveness (i.e., unconsciousness), and is only silenced at higher concentrations that induce deeper levels of anesthesia. We suggest that this persistent ACC activity underlies the phenomena of connected consciousness during general anesthesia, where otherwise anesthetized patients regain awareness intraoperatively and are capable of experiencing pain. As such, blocking the pain experience during general anesthesia likely occurs after isoflurane-induced silencing of the ACC, which we propose produces a similar functional outcome to ablating the ACC, a treatment known to provide pain relief.
In addition, we detail effective strategies for using the Zeiss LSM980 with Airyscan 2 to perform in vivo calcium imaging in awake and anesthetized mice. We also demonstrate how coupling Airyscan live imaging with chronically implanted gradient index (GRIN) lenses allows for identifying neural activity within distinct subpopulations of neurons. Lastly, we discuss methods for processing calcium imaging data to extract neural activity at the single neuron level.

Presented by: Naomi Kamasawa, Ph.D., Head of the Imaging Center at Max Planck Florida Institute for Neuroscience
Aubrey Funke, MS, Product Marketing Manager, Life Science EM & XRM, Carl Zeiss Microscopy

Neurons are three-dimensionally interconnected via numerous synapses in brain networks. Neuronal activity can be visualized and recorded with advanced light microscopy (LM) techniques, however, the resolution of electron microscopy (EM) is required to examine the ultrastructure of the synapse. In this talk, Dr. Naomi Kamasawa will introduce two workflows that her lab developed using correlative light and electron microscopy (CLEM) with volume EM techniques to link the function of neurons to synaptic ultrastructure: 1) functional properties of dendritic spines were captured with live 2-photon Ca2+ imaging, and the structural characteristics of the same dendrites and spines were analyzed using serial block-face scanning EM (SEM). We found a non-Hebbian synaptic weight distribution. 2) structural plasticity was induced onto single spines using 2-photon glutamate uncaging, and the ultrastructure of the exact same spines among other unstimulated spines was analyzed with serial-section array tomography SEM. We found a unique expansion of the peri-synaptic membrane at early point of the plasticity. At the end of the talk, join Aubrey Funke to learn about the new volume EM solution from ZEISS. ZEISS Volutome is the next generation of serial block-face imaging: highly automated, fully integrated and optimized for volume data creation. Discover the features that save you time while allowing you to access large volume data with superb image quality at low kV.