Fig 2: VIP+ INs innervate distinct populations of cells labeled in GAD65-EGFP knock-in mice. A, An image taken from the amygdala region of a GAD65-EGFP mouse. There is a clear difference in the quantity of EGFP-expressing cells between the BLA and striatal-like amygdala regions (CeA, central amygdala; Astr, amygdalostriatal area). B, CCK, NPY, VIP, CR, and CaMKII, but not PV, were found to be expressed in EGFP+ neurons. White arrows show green cells that express the given neurochemical markers, white arrowheads indicate green cells that do not contain the given neurochemical markers, and open arrowheads point to PV-containing cells that were not EGFP+. C, Pie charts show the percentage of EGFP+ neurons containing the given neurochemical markers in both LA and BA nuclei. The “Others” category comprises all green cells that lack the immunoreactivity for the neurochemical markers tested and the three SOM+/EGFP+ neurons. As VIP and CR form partially overlapping groups of INs, they were pooled together. D, Maximum projection intensity images of representative biocytin-filled neurons from four cell types expressing EGFP. Insets show their characteristic responses upon step current injections. CCKBC, CCK+ basket cell; NGF, neurogliaform cell; PN, principal neuron. Below each image, the postsynaptic currents evoked by whole-field blue-light illumination with high chloride intrapipette solution are shown. Blue arrows show the beginning of a 10-ms-long light illumination. Gray traces indicate five consecutive events, while the black traces are their averages. E, Bar graph shows the percentage of different types of EGFP+ neurons in which blue light-evoked postsynaptic events could be detected. F, Plot shows the magnitude of inhibitory postsynaptic responses recorded in different types of EGFP+ neurons (CCK-BC, 0.77 ± 0.21 nS, n = 6; NGF, 0.41 ± 0.11 nS, n = 7; IS-IN, 0.66 ± 0.14 nS, n = 12; PN, 0.89 ± 0.54 nS, n = 7). Each dot represents the average peak conductance (IPSG) obtained in individual cells, while horizontal bars indicate the mean. Scale bars: A, 150 µm; B, 20 µm; D, top, 40 µm (inset: x = 100 ms, y = 20 mV); D, bottom, x = 5 ms, y = 10 pA).

Fig 3: In vivo reprogramming of reactive hippocampal glia into GABAergic iNs in adult MTLE-HS mice(A) Experimental procedures.(B) DSRED+ hippocampal glia transduced with the control retrovirus (DsRed), 6 wpi. None of the DSRED+ cells express NEUN.(C) Proportion of control-transduced cells (DSRED) expressing glial (OLIG2, GFAP, or IBA1; n = 3) or neuronal marker (NEUN; n = 5), 6 wpi.(D–H) Hippocampal reactive glia transduced with the Ascl1/Dlx2-encoding retrovirus (DsRed) are reprogrammed into iNs.(D) DSRED+ iN expressing DCX, 7 dpi.(E) Proportion of DSRED+ cells expressing DCX or NEUN following expression of Ascl1/Dlx2 (DCX, n = 5; NEUN, n = 6) or control (n = 3), 7 dpi.(F) Ascl1/Dlx2-iNs (DSRED, white) exhibit complex neuronal morphologies and extend fibers creating dense networks throughout the dentate gyrus, 6 wpi.(G) Magnified views of the area boxed in (F) showing that Ascl1/Dlx2-iNs (DSRED) express NEUN (arrowheads), 6 wpi.(H) Proportion of DSRED+ cells converted into NEUN+ iNs following expression of Ascl1/Dlx2 (n = 6) or control (n = 5; same mice as in C), 6 wpi.(I–K) BrdU labeling of iNs.(I) Experimental procedures.(J) DSRED/NEUN+ iNs labeled by BrdU (arrowheads, drinking water protocol), 6 wpi.(K) Proportion of DSRED/NEUN+ iNs immunoreactive for BrdU following single BrdU pulse (n = 4) or BrdU supply in drinking water (n = 4), 6 wpi.(L–N) Conversion of hippocampal reactive glia into GABAergic iNs in GAD67-GFP mice.(L) Experimental procedures.(M) Ascl1/Dlx2-iNs (DSRED) expressing GFP (arrowheads; 6 wpi) demonstrating their GABAergic identity.(N) Proportion of DSRED/NEUN+ iNs expressing GFP following Ascl1/Dlx2 expression (n = 5), 6 wpi.(O) Ascl1/Dlx2-iNs (DSRED) express GAD67.(P) Ascl1/Dlx2-iNs (DSRED) expressing VIP, SST, or NPY (full arrowheads). Empty arrowhead points to a VIP-negative iN.(Q) Proportion of DSRED/NEUN+ iNs expressing VIP (n = 5), SST (n = 7), or NPY (n = 5), 8 wpi.Bars, mean ± SEM. Statistical analysis (C, E, and H): two-tailed Mann Whitney test. *p < 0.05, **p < 0.01.Right (B and J) and bottom (G) panels: magnified views of boxed areas. Composite images, (B), (F), and (G).Scale bars: 10 µm except 25 µm (B and F).See also Figure S3.