Cre Adeno-associated virus(AAV Serotype 1)

Astrocytes exhibit localized Ca²⁺ microdomain (MD) activity that is thought to be actively involved in information processing in the brain. However, little is known about the functional organization of Ca²⁺ MDs in space and time in relation to behavior and neuronal activity. Here, researchers first show that adeno-associated viral (AAV) particles are transferred anterogradely from axons to astrocytes. They then use this axo-astrocyte AAV transfer to express a genetically encoded Ca²⁺ indicator in high-contrast circuits specifically. Combining two-photon microscopy and unbiased, event-based analysis, cortical astrocytes embedded in vibrissa-thalamocortical circuits were studied. Researchers found widespread Ca²⁺ MD signals, some of which were ultrafast (≤300 ms). The frequency and magnitude of the signals increased substantially in response to movement but only modestly in response to sensory stimulation. The overlay of these signals yielded behavior-related maps with characteristic hotspots of Ca²⁺ activity that may represent memory engrams. These functional subdomains were stable over days, suggesting subcellular specialization.

Here, the researchers first tested whether injection of AAV1 in the VPM would lead to transduction of BX astrocytes and neurons (Figure 1A). They co-injected AAV1 delivering the Cre gene under the cytomegalovirus promoter (CMV) AAV1-CMV-Cre and AAV1 delivering the red chromophore TurboRFP gene under the human synapsin promoter (hSyn) AAV1-hSyn-TurboRFP in the VPM. If small amounts of AAV were transported anterogradely, ubiquitous, CMV promoter-driven Cre recombinase expression in BX astrocytes and neurons would be expected, which in turn could activate a flip-out excision (FLEx) switch to drive conditional gene expression. AAV1-hSyn-TurboRFP was used to label thalamocortical axonal projections.

Figure 1. AAV1 transfer from VPM thalamocortical neurons to BX astrocytes and neurons. (Georgiou L, et al., 2022)

To test whether Cre is present in BX astrocytes, we also injected AAV5-GFaABC1D-FLEx-lck-GCaMP6f in the BX to induce Cre-dependent GECI GCaMP6f expression selectively in the astrocyte plasma membrane (Figure 1B). This intersectional approach resulted in sparse labeling of L2/3 BX astrocytes 3 weeks after AAV injection (Figure 1B). The GFaABC1D promoter induces astrocyte-specific gene expression, the FLEx system allows conditional expression in the few astrocytes expressing Cre recombinase, and the lck tag leads to membrane labeling of GCaMP6f, revealing the cloud-like morphology of astrocyte nanoscale processes (Figure 1B). TurboRFP labeled thalamocortical axonal projections to reveal the typical barrel-shaped projection pattern expected in L4 of the BX (Figure 1B and C, middle).