Isoflurane exposure leads to aberrant activation of the mechanistic target of rapamycin (mTOR) signaling pathway, and pharmacological inhibition of the mTOR activities rescues deficits in behavioral tests and loss of spines.

<p>(A) Representative confocal images of phospho-S6 (pS6) immunofluorescence at postnatal day (P) 30 in the dentate gyrus showing an increase in labeling in the isoflurane plus vehicle (Iso/V) group relative to controls and a return to baseline in the group exposed to isoflurane and subsequently treated with rapamycin, designated Iso/R. The upper panels are original confocal images with DAPI in blue and pS6 labeling in red, and the lower panels are processed for quantification with black pS6 signal on white background (ML, molecular layer; DG, dentate gyrus; HI, hilus, scale bar: 50 μm). Also shown in (A) quantification of normalized pS6 expression in the dentate gyrus granule cell layer (***<i>p</i> < 0.001, ANOVA, numbers in each bar represent n for images analyzed). (B) Schematic diagram of rapamycin treatment for behavior tests and spine analysis. Summaries of total dendritic length (C) and Sholl analysis of dendritic complexity (D) of GFP+ neurons show a rescue of normal dendritic arbor length and complexity with Iso/R. Values represent mean ± SEM (*<i>p</i> < 0.05, **<i>p</i> < 0.01; ANOVA for C; *<i>p</i> < 0.0001 ANOVA for D). Numbers in each bar represent number of cells analyzed per group, minimum of 5 animals per group). Summaries of object-place recognition test (E) and Y-maze test (F) for Iso/V and Iso/R show a recovery to near control performance with Iso/R. (Control <i>n</i> = 10, Iso/V <i>n</i> = 11, Iso/R <i>n</i> = 11; *: <i>p</i> < 0.05; **: <i>p</i> < 0.01, Student <i>t</i> test). (G) Representative confocal images of dendritic spines at P60. Scale bar: 2 μm. Shown on right are summary plots of total and mature dendritic spine density. Numbers associated with bar graph indicate the number of dendritic segments examined, a total of 2,586 spines in the control group, 1,831 spines in the isoflurane plus vehicle group, and 2,999 spines in the isoflurane plus rapamycin group were analyzed (****<i>p</i> < 0.0001; ns: non-significant; ANOVA, numbers in each bar represent n of dendritic segments analyzed per group, minimum of 5 animals per group). Underlying data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001246#pbio.2001246.s008" target="_blank">S1 Data</a> under Fig 3A-G.</p

Isoflurane exposure results in overgrowth of dendritic arbors.

<p>(A) A schematic diagram of isoflurane exposure procedure for morphology examination. (B) Sample confocal image of dentate gyrus granule cell (DGCs) infected with retrovirus expressing green florescent protein (GFP) (scale bar: 100 μm). Representative confocal images (C) and tracings (D) of individual control and isoflurane-exposed GFP+ neurons at postnatal day (P) 30 exhibiting overgrowth in the isoflurane group relative to control conditions (scale bar: 10 μm for both C and D). Summaries of total dendritic length (E) and Sholl analysis of dendritic complexity (F) of GFP+ neurons show marked overgrowth of dendritic arbors. Numbers associated with bar graph indicate the number of neurons examined from at least 5 animals per group. The same groups of neurons were examined in (E) and (F). Values represent mean ± SEM (**<i>p</i> < 0.01; Student <i>t</i> test for E and *<i>p</i> < 0.0001 ANOVA for F). Underlying data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001246#pbio.2001246.s008" target="_blank">S1 Data</a> under Fig 1F tab.</p