XI European Meeting on Glial Cell Function in Health and Disease

Overview Session Overview Sessionprint print  

July 3, 2013 - Hall A 9:00am - 10:55am
Workshop I: Imaging of Glial Cell Activity

Organized by
Hajime Hirase (RIKEN Brain Science Institute, Wako, Japan)
Frank Kirchhoff (University of Saarland, Homburg, Germany)


Workshop I: Imaging of Glial Cell Activity - Introduction

*Frank  Kirchhoff 1
1 University of Saarland, Institute of Physiology, Department of Molecular Physiology, Homburg, Germany
Abstract text :

During the last few years imaging of neuron-glia interactions was facilitated tremendously by the development of genetically encoded fluorescent dyes as well as by improved microscopy techniques. In this workshop, the latest genetic tools and microscopic approaches will be presented and discussed, thereby highlighting the important roles of glial cells for brain function.


Functional imaging of neuro-glio-vascular network activity

*Yuji Ikegaya 1
1 University of Tokyo, , Bunkyo-ku, Japan
Abstract text :

Astrocytes exhibit various patterns of intracellular calcium elevations, which are often involved in gliotransmission. Although local synchronization and wave-like propagation of astrocytic calcium activities are often reported, the temporal activity patterns of individual cells are poorly characterized at a larger-scale and longer-time scale. Here, I utilized in vivo and in vitro calcium imaging technique to simultaneously visualize and analyze the activity patterns of hundreds of astrocytes from mouse cortex and hippocampus and found that among the observed astrocytes, 24.9 ± 17.1% cells (mean ± SD) showed unique oscillation patterns, which I named "intermittent oscillations". These cells repeatedly showed clustered oscillation events at an interval of 859 ± 661 s, with only few sporadic activities between the events. Single events persisted for 107.5 ± 56.4 s and involved 3.9 ± 1.6 cycles. At larger-scale dynamics in vivo, astrocytes often exhibited synchronized activity among neighboring cells. The astrocytic synchronization was more frequent in awake mice than in anesthetized ones and was often triggered by sensory stimulation and systemic treatment with adrenergic agonist. Further properties, pharmacological profiles, and spatial allocations of these cells are now under investigation. 


Astrocytic Ca2+ surges, gamma oscillations, and synaptic plasticity

*Hajime Hirase 1
1 RIKEN, , Wako, Japan
Abstract text :

Global brain state dynamics regulate plasticity in local cortical circuits but the underlying cellular and molecular mechanisms are unclear. Here, we demonstrate that astrocyte Ca2 signaling provides a critical bridge between cholinergic activation, associated with attention and vigilance states, and somatosensory plasticity in mouse barrel cortex in vivo. We investigate first that a combined stimulation of mouse whiskers and the nucleus basalis of Meynert (NBM), the principal source of cholinergic innervation to the cortex, leads to enhanced whisker-evoked field potential response. This plasticity is dependent on muscarinic receptors (mAChR) and NMDAR. During the induction of this synaptic plasticity, we find that astrocytic [Ca2 ]i is pronouncedly elevated, which is blocked by mAChR antagonists. The elevation of astrocytic [Ca2 ]i is crucial in this type of synaptic plasticity, as the plasticity could not be induced in inositol 1,4,5-trisphosphate receptor type 2 knockout (IP3R2-KO) mice, in which astrocytic [Ca2 ]i surges are diminished. Moreover, NBM stimulation led to a significant increase in the extracellular concentration of the NMDAR coagonist D-serine in wild type mice, but not in IP3R2-KO mice. Finally, plasticity in IP3R2-KO mice could be rescued by externally supplying D-serine. Our data present coherent lines of in vivo evidence for astrocytic involvement in cortical plasticity. On the other hand, our preliminary experiments show that both wild type and IP3R2 knockout mice exhibit similar NBM-triggered cerebral cortical blood flow changes.


Imaging Ca2+ transients in astrocyte microdomains using the genetically encoded indicator GCaMP3

*Amit Agarwal 1 , Ethan  Hughes 1 , Dwight  Bergles 1
1 Johns Hopkins University, , Baltimore, United States
Abstract text :

Astrocytes elevate intracellular Ca2 in response to stimulation of metabotropic receptors. These Ca2 transients are linked to processes as diverse as synaptic plasticity and functional hyperemia, but the relevance of this signaling remains uncertain. Although most studies of Ca2 signaling in astrocytes have focused on somatic events, anatomical and functional studies indicate that Ca2 signaling in astrocytes may be compartmentalized into functionally isolated “microdomains.” To facilitate analysis of Ca2 signaling, we generated transgenic mice in which the cytosolic and membrane anchored variant of genetically encoded calcium indicator GCaMP3, referred as cGCaMP3 and mGCaMP3 respectively, can be expressed conditionally in a cell specific manner. A ubiquitous CAG promoter is used to control cGCaMP3 and mGCaMP3 expression, and a “stopper” sequence flanked by loxP sites was placed upstream of the coding sequence, preventing expression until Cre excises this DNA. These constructs were targeted to ROSA26 locus to ensure widespread expression. To evaluate whether cGCaMP3 and mGCaMP3 were expressed at levels sufficient to resolve Ca2 transients, we bred R26-lsl-cGCaMP3 and R26-lsl-mGCaMP3 mice to GLAST-CreER mice. Double transgenic offspring were injected with tamoxifen at 3 weeks of age and analyzed 2-3 weeks later. Histology revealed that cGCaMP3 and mGCaMP3 were expressed in astrocytes throughout the brain. Astrocytes in acute cortical slices exhibited large amplitude, spontaneous increases in both cGCaMP3 and mGCaMP3 fluorescence. The signal to noise ratio of the fluorescence intensity was greatly improved in astyrocytes expressing mGCaMP3, partially due to the ability of mGCaMP3 to better resolve near membrane Ca2 flux. Ca2 transients were typically restricted to small regions of an individual astrocyte, consistent with Ca2 elevations within microdomains. Spontaneous Ca2 transients were occasionally observed in the soma of astrocytes expressing cGCaMP3 but not mGCaMP3, and were not coincident with activity in the processes, suggesting that these regions are functionally uncoupled. To determine if GCaMP3 expression is sufficient to visualize Ca2 signaling in vivo, we made cranial windows over the somatosensory cortex and performed 2-photon imaging. Microdomain Ca2 transients were prominent in cortical astrocytes, indicating that these mice provide new opportunities for understanding the significance of this form of signaling.