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. 2012:2012:425818.
doi: 10.1155/2012/425818. Epub 2012 Oct 2.

Calcium imaging of living astrocytes in the mouse spinal cord following sensory stimulation

Giovanni Cirillo  1 , Daniele De Luca, Michele Papa
Affiliations

Calcium imaging of living astrocytes in the mouse spinal cord following sensory stimulation

Giovanni Cirillo et al. Neural Plast. 2012.

Abstract

Astrocytic Ca(2+) dynamics have been extensively studied in ex vivo models; however, the recent development of two-photon microscopy and astrocyte-specific labeling has allowed the study of Ca(2+) signaling in living central nervous system. Ca(2+) waves in astrocytes have been described in cultured cells and slice preparations, but evidence for astrocytic activation during sensory activity is lacking. There are currently few methods to image living spinal cord: breathing and heart-beating artifacts have impeded the widespread application of this technique. We here imaged the living spinal cord by two-photon microscopy in C57BL6/J mice. Through pressurized injection, we specifically loaded spinal astrocytes using the red fluorescent dye sulforhodamine 101 (SR101) and imaged astrocytic Ca(2+) levels with Oregon-Green BAPTA-1 (OGB). Then, we studied astrocytic Ca(2+) levels at rest and after right electrical hind paw stimulation. Sensory stimulation significantly increased astrocytic Ca(2+) levels within the superficial dorsal horn of the spinal cord compared to rest. In conclusion, in vivo morphofunctional imaging of living astrocytes in spinal cord revealed that astrocytes actively participate to sensory stimulation.

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Figures

Figure 1
Figure 1
Experimental setup. (a) Two-photon laser scanning microscope setup. (b) Schematic section of the lumbar spinal cord showing the regions of interest (laminae I-II). (c) Exposed spinal cord surface, after vertebral laminectomy, before SR101/OGB loading. In the middle line, the posterior medial spinal vein.
Figure 2
Figure 2
Selective labeling of astrocytes with SR101 and calcium indicator OGB. (a) A subset of SR101-labeled astrocytes in the superficial laminae of dorsal horn of lumbar spinal cord. (b) OGB-labeled astrocytes in the same ROI (scale bar: 20 μm).
Figure 3
Figure 3
Sensory stimulation triggers Ca2+ increase in spinal astrocytes. Section of superficial laminae of dorsal horn of lumbar spinal cord, showing two different astrocytes (a1, a2) in rest condition (a) and following sensory stimulation (b) (scale bar: 10 μm). (c) Quantitative analysis of astrocytic Ca2+ levels during rest and stimulation expressed as ΔF/F 0. The mean value of Ca2+ increase during stimulation was significantly higher than that measured during rest (rest versus stimulus, **P ≤ 0.001).
Figure 4
Figure 4
Time-lapse/z stack serial images of lumbar spinal cord. Spinal vessels are labeled with Dextran-Rhodamine; astrocytes adjacent to spinal vessels are labeled with OGB (scale bar: 10 μm).
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