Glutamate-dependent neuroglial calcium signaling differs between young and adult brain

Abstract

An extensive literature shows that astrocytes exhibit metabotropic glutamate receptor 5 (mGluR5)-dependent increases in cytosolic calcium ions (Ca(2+)) in response to glutamatergic transmission and, in turn, modulate neuronal activity by their Ca(2+)-dependent release of gliotransmitters. These findings, based on studies of young rodents, have led to the concept of the tripartite synapse, in which astrocytes actively participate in neurotransmission. Using genomic analysis, immunoelectron microscopy, and two-photon microscopy of astrocytic Ca(2+) signaling in vivo, we found that astrocytic expression of mGluR5 is developmentally regulated and is undetectable after postnatal week 3. In contrast, mGluR3, whose activation inhibits adenylate cyclase but not calcium signaling, was expressed in astrocytes at all developmental stages. Neuroglial signaling in the adult brain may therefore occur in a manner fundamentally distinct from that exhibited during development.

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.

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.

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 (ΔF/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 (ΔF/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.

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 (ΔF/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 (ΔF/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.