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. 2021 Jun 10;11:12242. doi: 10.1038/s41598-021-91770-7

Figure 2.

Figure 2

Adeno-associated virus (AAV)-mediated deletion of Per2 in glial cells of adult mice leads to a depression resistant behavior. (A) Fluorescent imaging of whole brains 3 weeks after no injection (left), intravenous (i.v.) injection of the engineered AAV-PHP.eB, which can pass the blood–brain barrier (BBB), containing the general CAG driver (second from left) or the glial Gfap driver (middle). The second to last brain is from an animal with i.v. injected AAV9, which does not pass the BBB, containing the general CAG driver. The last brain (right) is from an animal injected intraperitoneally (i.p.) with the AAV-PHP.eB Gfap-driven construct. Note that only the brains of animals that received the AAV-PHP.eB i.v. display significant fluorescent signal after 3 weeks (orange and yellow color). (B) Fluorescent imaging of whole brains 2 months after injection of the AAV-PHP.eB. Note that the fluorescence is still maintained after 2 months post injection and that even the i.p. injected AAV-PHP.eB Gfap is showing signal in the brain now. (C) Sorting of neurons and astrocytes by flow cytometry from brain tissue including the nucleus accumbens (NAc). The left panel shows the removal of debris from a single cell suspension, showing the distribution of debris in the forward as well as in the side scatter (FSC and SSC, respectively). The middle panel shows the removal of CD11b+ cells (microglia) from the cell suspension. The CD11b- cells (bottom half from middle panel) were then sorted into two distinct cell populations corresponding to astrocytes (GLT1+/CD90.2-) and neurons (CD90.2+/GLT1-) (right panel). (D) PCR analysis of astrocytes and neurons from the cell sorting. Microglia (CD11b+) as well as astrocytes (GLT1+/CD90.2-), but not neurons (CD90.2+/GLT1-) from PHP.eB Gfap-iCre infected animals express iCre, indicating that only glia and not neurons could express iCre in order to delete Per2 in the Per2fl/fl mice. All probes and markers were loaded on the same gel and the resulting photograph was not separated and recomposed. (E) Immunohistochemistry of vGPer2 brain tissue from nucleus accumbens (NAc) isolated at ZT6. The signal for PER2 (green) mainly overlaps with neuronal NeuN signal (red) giving rise to the yellow color. Scale bar: 100 µm. (F) Immobility time in the forced swim test (FST) of vGPer2 (PHP.eB Gfap-iCre, green) and control (PHP.eB control, blue) animals are shown (n = 7, two-tailed t-test, **P < 0.01). (G) Representative swimograms of vGPer2 (AAV-PHP.eB Gfap-iCre, green) and control (AAV-PHP.eB control, blue) animals. Note the longer stretches of immobility in the control animals (right panel, blue).