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New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes In Situ and In Vivo.

ABSTRACT: Astrocytes exist throughout the nervous system and are proposed to affect neural circuits and behavior. However, studying astrocytes has proven difficult because of the lack of tools permitting astrocyte-selective genetic manipulations. Here, we report the generation of Aldh1l1-Cre/ERT2 transgenic mice to selectively target astrocytes in vivo. We characterized Aldh1l1-Cre/ERT2 mice using imaging, immunohistochemistry, AAV-FLEX-GFP microinjections, and crosses to RiboTag, Ai95, and new Cre-dependent membrane-tethered Lck-GCaMP6f knockin mice that we also generated. Two to three weeks after tamoxifen induction, Aldh1l1-Cre/ERT2 selectively targeted essentially all adult (P80) brain astrocytes with no detectable neuronal contamination, resulting in expression of cytosolic and Lck-GCaMP6f, and permitting subcellular astrocyte calcium imaging during startle responses in vivo. Crosses with RiboTag mice allowed sequencing of actively translated mRNAs and determination of the adult cortical astrocyte transcriptome. Thus, we provide well-characterized, easy-to-use resources with which to selectively study astrocytes in situ and in vivo in multiple experimental scenarios.

SUBMITTER: Srinivasan R

PROVIDER: S-EPMC5403514 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes In Situ and In Vivo.

Srinivasan Rahul R Lu Tsai-Yi TY Chai Hua H Xu Ji J Huang Ben S BS Golshani Peyman P Coppola Giovanni G Khakh Baljit S BS

Neuron 20161208 6

Astrocytes exist throughout the nervous system and are proposed to affect neural circuits and behavior. However, studying astrocytes has proven difficult because of the lack of tools permitting astrocyte-selective genetic manipulations. Here, we report the generation of Aldh1l1-Cre/ERT2 transgenic mice to selectively target astrocytes in vivo. We characterized Aldh1l1-Cre/ERT2 mice using imaging, immunohistochemistry, AAV-FLEX-GFP microinjections, and crosses to RiboTag, Ai95, and new Cre-depend ...[more]

PMID: 27939582

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Project description:We recently showed that inhibition of neuronal activity, glutamate uptake, or reversed-Na(+)/Ca(2+)-exchange with TTX, TFB-TBOA, or YM-244769, respectively, increases mitochondrial mobility in astrocytic processes. In the present study, we examined the interrelationships between mitochondrial mobility and Ca(2+) signaling in astrocyte processes in organotypic cultures of rat hippocampus. All of the treatments that increase mitochondrial mobility decreased basal Ca(2+). As recently reported, we observed spontaneous Ca(2+) spikes with half-lives of ?1 s that spread ?6 ?m and are almost abolished by a TRPA1 channel antagonist. Virtually all of these Ca(2+) spikes overlap mitochondria (98%), and 62% of mitochondria are overlapped by these spikes. Although tetrodotoxin, TFB-TBOA, or YM-244769 increased Ca(2+) signaling, the specific effects on peak, decay time, and/or frequency were different. To more specifically manipulate mitochondrial mobility, we explored the effects of Miro motor adaptor proteins. We show that Miro1 and Miro2 are both expressed in astrocytes and that exogenous expression of Ca(2+)-insensitive Miro mutants (KK) nearly doubles the percentage of mobile mitochondria. Expression of Miro1(KK) had a modest effect on the frequency of these Ca(2+) spikes but nearly doubled the decay half-life. The mitochondrial proton ionophore, FCCP, caused a large, prolonged increase in cytosolic Ca(2+) followed by an increase in the decay time and the spread of the spontaneous Ca(2+) spikes. Photo-ablation of mitochondria in individual astrocyte processes has similar effects on Ca(2+). Together, these studies show that Ca(2+) regulates mitochondrial mobility, and mitochondria in turn regulate Ca(2+) signals in astrocyte processes.In neurons, the movement and positioning of mitochondria at sites of elevated activity are important for matching local energy and Ca(2+) buffering capacity. Previously, we demonstrated that mitochondria are immobilized in astrocytes in response to neuronal activity and glutamate uptake. Here, we demonstrate a mechanism by which mitochondria are immobilized in astrocytes subsequent to increases in intracellular [Ca(2+)] and provide evidence that mitochondria contribute to the compartmentalization of spontaneous Ca(2+) signals in astrocyte processes. Immobilization of mitochondria at sites of glutamate uptake in astrocyte processes provides a mechanism to coordinate increases in activity with increases in mitochondrial metabolism.

Project description:BackgroundAccumulating evidence suggests that glial signalling is activated by different brain functions. However, knowledge regarding molecular mechanisms of activation or their relation to neuronal activity is limited. The purpose of the present study is to identify the characteristics of ATP-evoked glial signalling in the brain reward area, the nucleus accumbens (NAc), and thereby to explore the action of citric acid cycle intermediate succinate (SUC).ResultsWe described the burst-like propagation of Ca2+ transients evoked by ATP in acute NAc slices from rat brain. Co-localization of the ATP-evoked Ca2+ signalling with immunoreactivities of the astroglia-specific gap junction forming channel protein connexin43 (Cx43) and the glial fibrillary acidic protein (GFAP) indicated that the responsive cells were a subpopulation of Cx43 and GFAP immunoreactive astrocytes. The ATP-evoked Ca2+ transients were present under the blockade of neuronal activity, but were inhibited by Ca2+ store depletion and antagonism of the G protein coupled purinergic P2Y1 receptor subtype-specific antagonist MRS2179. Similarly, Ca2+ transients evoked by the P2Y1 receptor subtype-specific agonist 2-(Methylthio)adenosine 5'-diphosphate were also blocked by MRS2179. These characteristics implied that intercellular Ca2+ signalling originated from the release of Ca2+ from internal stores, triggered by the activation of P2Y1 receptors. Inhibition by the gap junction blockers carbenoxolone and flufenamic acid and by an antibody raised against the gating-associated segment of Cx43 suggested that intercellular Ca2+ signalling proceeded through gap junctions. We demonstrated for the first time that extracellular SUC also evoked Ca2+ transients (EC50 = 50-60 ?M) in about 15% of the ATP-responsive NAc astrocytes. By contrast to glial cells, electrophysiologically identified NAc neurons surrounded by ATP-responsive astrocytes were not activated simultaneously.ConclusionsWe concluded, therefore, that ATP- and SUC-sensitive Ca2+ transients appear to represent a signalling layer independent of NAc neurons. This previously unrecognised glial action of SUC, a major cellular energy metabolite, may play a role in linking metabolism to Ca2+ signalling in astrocytic networks under physiological and pathological conditions such as exercise and metabolic diseases.

Dataset Information

New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes In Situ and In Vivo.

Publications

New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes In Situ and In Vivo.

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