Astrocyte morphology determines properties of the astroglial networks

Abstract

Astrocytes are very abundant glial cells in the brain. They perform many functions: provision of nutrient to the neurons, maintenance of extracellular ion balance, shaping of network operations and synaptic signaling and plasticity. Astrocytes can form large heterogeneous networks throughout the brain. In the hippocampal CA1 region, astrocytes are connected by GJs that are mainly composed of GJα1 (Cx43) and GJβ6 (Cx30). Together, these proteins form hexameric structures called connexons which can connect two adjacent cells by creating GJs. The astroglial GJ coupling is important for various processes such as i) distribution of metabolites, ii) regulation of synaptic transmission and iii) plasticity and biochemical support to the endothelial cells which form the BBB. These networks were proposed to counteract hyperactivity by facilitating glutamate removal, thereby attenuating synaptic transmission. Moreover, coupled astrocyte networks are limited by the presence of the CA1 pyramidal cell layer which works as an anatomical barrier. This layer also impacts on the diffusion of astrocytes within the network they form. <br /> Previous experiments performed in the laboratory of Prof. Dr. Henneberger showed that induction of LTP or epileptiform activity rapidly changes the morphology of astrocyte and the diffusion within and between astrocytes in acute hippocampal slices. These changes may modify the tortuosity of intracellular space affecting diffusion of molecules within one cell and to neighboring ones. Therefore, the aim of this thesis was to investigate the fundamental relationship between astrocytic structure and the strength of astroglial coupling using combinations of 2-photon excitation fluorescence microscopy and electrophysiology. <br /> This study found that the primary morphological determinant of coupling strength is the complexity of the astroglial branching pattern (segmentation) rather than the total intracellular volume available for diffusion (VF). Using the FRAP technique, this project could also reveal that changes of intracellular diffusivity do not explain the link between astroglial morphological complexity and coupling. Moreover, this study also showed, by analyzing the location and density of Cx30 and Cx43, that the astrocyte structure does not define the pattern of GJ formation between astrocytes. Finally, astrocyte morphology was specifically manipulated by injecting two recombinant viruses in mice dorsal hippocampi. These viruses were able to affect the actin polymerization by altering the activity of RhoA GTPase and LIMK. The over-expression of the mutant variants of RhoA but not the LIMK inhibitor S3 peptide, resulted in a significant reduction of the astrocytic VF while both constructs were able to reduce the coupling strength in the glial coupled network. <br /> By a controlled manipulation of the astrocyte cytoskeleton rearrangement via viral injections this study was able to show how glial morphology shapes connections within the astrocyte network. This provides the basis for further studies of network diffusivity in both physiological and pathophysiological conditions.