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Science 339 (6116): 197-200

Copyright © 2013 by the American Association for the Advancement of Science

Glutamate-Dependent Neuroglial Calcium Signaling Differs Between Young and Adult Brain

Wei Sun1,*, Evan McConnell1,*, Jean-Francois Pare2,*, Qiwu Xu1,*, Michael Chen1, Weiguo Peng1, Ditte Lovatt1, Xiaoning Han1, Yoland Smith2, and Maiken Nedergaard1,{dagger}

1 Division of Glial Disease and Therapeutics, Center for Translational Neuromedicine, University of Rochester, Rochester, NY 14642, USA.
2 Yerkes National Primate Research Center and Department of Neurology, Emory University, 954 Gatewood Road NE, Atlanta, GA 30329, USA.


Figure 1
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Fig. 1. Expression profile of mGluRs in human and mouse astrocytes. (A to C) Microarray analysis of the expression of mGluR in (A) adult human cortical astrocytes, (B) adult mouse cortical astrocytes, and (C) adult mouse hippocampal astrocytes. Grm3 is the major mGluR expressed by human cortical astrocytes and mouse cortical and hippocampal astrocytes. (D) qPCR analysis of GLT1+ populations confirmed that GRM3 is highly enriched in human cortical astrocytes. (E) qPCR analysis of GLT1+ cell populations showed that Grm3 also is highly enriched in mouse cortical astrocytes. (F) Expression of Grm3 and Grm5 in mouse hippocampal astrocytes isolated from 1-, 2-, 3-, and 12-week-old mice. Error bars, mean ± SEM; n = 3 biological samples. *P < 0.05, one-way ANOVA, Bonferroni multiple comparisons test.

 

Figure 2
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Fig. 2. Electron micrographic analysis of mGluR2/3 and mGluR5 in the adult mouse cortex and hippocampus. (A to D) Electron micrographs showing examples of mGluR5-immunoreactive elements, and (E to G) mGluR2/3-immunoreactive elements in the mouse hippocampus (Hipp) and cerebral cortex (Ctx). Ax, unmyelinated axons; Den, dendrites; As, astrocytes; Te, axon terminals. Scale bars, 0.5 μm. (H and I) Histograms showing the relative percentage of mGluR5- or mGluR2/3-immunoreactive elements categorized as presynaptic (terminals and unmyelinated axons) or postsynaptic (dendrites and spines) neuronal elements or glia. For each region and antibody, data were collected in three mice. A total of 2215 μm2 of mGluR5 or mGluR2/3 immunostained tissue was examined in both cortex and hippocampus.

 

Figure 3
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Fig. 3. mGluR5 agonists trigger astrocytic Ca2+ signaling in slices prepared from mice pups. (A) The left panel shows enhanced green fluorescent protein (eGFP) driven by the GLT1 promoter; the three right panels depict relative increase in rhod-2 fluorescence signal ({triangleup}F/F) in response to microinjection of t-ACPD in a hippocampal slice prepared from a 15-day-old mouse pup. The injection pipette is outlined. Green circle represents Alexa 488 wavefront (not shown). White arrows indicate astrocytes displaying an increase in Ca2+. (B) aCSF injection in a hippocampal slice. (Top) Changes in rhod-2 fluorescence emission ({triangleup}F/F); (bottom) Alexa 488 diffusion. (C to E) Histograms comparing (C) number of responding astrocytes, (D) Ca2+ amplitude, and (E) Ca2+ wave velocity and Alexa 488 diffusion velocity (internal control for consistency of agonist injection). ***P < 0.001, one-way ANOVA, Bonferroni test; n = 8 to 9 trials.

 

Figure 4
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Fig. 4. Multiple mGluR5 agonists fail to trigger astrocytic Ca2+ signaling in adult mice. (A) Experimental setup used for microinjection of agonist in cerebral cortex layer II in anesthetized mice loaded with rhod-2 AM. (B) (Top left) EGFP driven by the astrocyte-specific GLT1 promoter in an adult mouse. (Top right) Time lapse of changes in rhod-2 fluorescence signal ({triangleup}F/F) in response to microinjection of t-ACPD (500 μM). One astrocyte (white arrow) exhibited an increase in rhod-2 signal. (Bottom) Rhod-2 signal changes ({triangleup}F/F) in response to ATP (500 μM) in an adult mouse. White arrows indicate astrocytes that exhibited an increase in Ca2+. (C to E) Histograms comparing (C) number of astrocytes displaying an increase in Ca2+ in response to agonist injections in pups and adult mice, (D) Ca2+ amplitude, and (E) Ca2+ wave velocity and Alexa 488 diffusion velocity (internal control for consistency of agonist injection). **P < 0.01, ***P < 0.001, one-way ANOVA, Bonferroni test; n = 4 to 19 trials.

 


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