Transgenic mice expressing a cameleon fluorescent Ca2+ indicator in astrocytes and Schwann cells allow study of glial cell Ca2+ signals in situ and in vivo

Abstract

Glial cell Ca2+ signals play a key role in glial-neuronal and glial-glial network communication. Numerous studies have thus far utilized cell-permeant and injected Ca2+ indicator dyes to investigate glial Ca2+ signals in vitro and in situ. Genetically encoded fluorescent Ca2+ indicators have emerged as novel probes for investigating cellular Ca2+ signals. We have expressed one such indicator protein, the YC 3.60 cameleon, under the control of the S100β promoter and directed its expression predominantly in astrocytes and Schwann cells. Expression of YC 3.60 extended into the entire cellular cytoplasmic compartment and the fine terminal processes of protoplasmic astrocytes and Schwann cell Cajal bands. In the brain, all the cells known to express S100β in the adult or during development, expressed YC 3.60. While expression was most extensive in astrocytes, other glial cell types that express S100β, such as NG2 and CNP-positive oligodendrocyte progenitor cells (OP cells), microglia, and some of the large motor neurons in the brain stem, also contained YC 3.60 fluorescence. Using a variety of known in situ and in vivo assays, we found that stimuli known to elicit Ca2+ signals in astrocytes caused substantial and rapid Ca2+ signals in the YC 3.60-expressing astrocytes. In addition, forepaw stimulation while imaging astrocytes through a cranial window in the somatosensory cortex in live mice, revealed robust evoked and spontaneous Ca2+ signals. These results, for the first time, show that genetically encoded reporter is capable of recording activity-dependent Ca2+ signals in the astrocyte processes, and networks.

Long-term In Vivo Calcium Imaging of Astrocytes Reveals Distinct Cellular Compartment Responses to Sensory Stimulation

“…Early GECIs, such as Yellow Cameleon 3.60 and GCaMP3, have modest signal to noise ratios and relatively low affinity for calcium, which limited detection of spontaneous calcium microdomains within ramified astrocyte processes (Atkin et al 2009;Paukert et al 2014). High affinity genetic calcium indicators, such as Yellow Cameleon Nano50, detect long (~70 s) spontaneous calcium signals within astrocyte processes (Kanemaru et al 2014), which may reflect saturation of this sensor, making it difficult to elucidate individual peaks of different types.…”

Live Imaging of Protein Kinase Activities in Transgenic Mice Expressing FRET Biosensors

“…A high level of body fluorescence was detected in three of them, of which two showed heterogeneous expression, leaving only one transgenic mouse for the analysis. In many studies that reported the generation of transgenic mice expressing FRET biosensors, analysis was conducted in vitro with cells isolated from the tissues of transgenic mice (Atkin et al, 2009;Hara et al, 2004;Isotani et al, 2004;Nikolaev et al, 2006;Tsujino et al, 2005), and only a few of them succeeded in FRET imaging in living tissues (Atkin et al, 2009;Tsujino et al, 2005), probably due to low levels of expression. Recently, Yamaguchi et al succeeded in the generation of transgenic mice expressing a FRET biosensor for Caspase3 by introducing insulator sequences (Yamaguchi et al, 2011).…”

Astrocyte Calcium Signaling: From Observations to Functions and the Challenges Therein

“…Building on work with fluorescence resonance energy transfer based Ca 2þ indicators expressed in astrocytes (Atkin et al 2009;Russell 2011 indicators (GECIs) based on circularly permuted green fluorescent protein (Hires et al 2008;Tian et al 2009). We focused on using a membrane-targeted GECI and a cytosolic form (Fig.…”