Shh signaling activity is increased upon seizure induction.

(A) Representative Gli1-nLacZ staining images of the SGZ of Gli1nLacZ/+ mice 1, 3, or 7 days after KA-induced seizure. Control mice did not receive KA treatment. (B) Quantification of Gli1-LacZ+ cells in SGZ. Values represent mean ± SEM; ns: P > 0.05, **P < 0.01 One-way ANOVA with Tukey’s multiple comparison test. (C) Representative Gli1-LacZ staining from SGZ of Gli1nLacZ/+ and Shh+/-; Gli1nLacZ/+mice 3 days after KA-induced seizure. (D) Quantification of Gli1-nLacZ+ cells in SGZ without (Naive) and with seizure induction (KA). Values represent mean ± SEM; ns: P > 0.05, **P < 0.01, ****P < 0.0001. Two-way ANOVA with Tukey’s multiple comparison test. (E) Experimental scheme of analyzing neurogenesis by BrdU pulse labeling. Seizures were induced in two-month-old (2m) mice by KA injection. One day after KA-induced seizures, the mice received BrdU for 5 days and then were analyzed 3 days after the last BrdU injection. (F) Representative immunofluorescence images of newborn neurons labeled with BrdU (green), DCX (red) and DAPI (blue) in the SGZ of wild-type (WT) and Shh+/- mice after seizure induction. (G) Higher magnification images from inset of panel (F), representing ectopic neurons in the hilus, which are indicated by white arrowheads. (H and I) Quantification of newborn neurons (H) and ectopic neurons (I) produced after seizure induction in the SGZ of WT and Shh+/- mice. Values represent mean ± SEM; ns: P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Two-way ANOVA with Tukey’s multiple comparison test. #P < 0.05. Unpaired t-test (two-tailed) in two groups (WT vs Shh+/- in naive condition). There was reduction of DCX+ BrdU+ cells between WT vs Shh+/- in naive condition.

Shh is expressed in mossy cells and upregulated upon seizure induction.

(A) Representative immunofluorescence images of mossy cells labeled with GluR2/3 (green) and ShhCre/+;Ai14 (tdTomato; red) 1 week after seizure induction. (B) Quantification of tdTomato+ cells in each area of the DG. GCL; granule cell layer, ML; molecular layer. (C) Representative immunofluorescence images of interneurons labeled with parvalbumin (cyan) and ShhCre/+;Ai14 (tdTomato; red) in the hilus. (D) Cell-type population of tdTomato+ cells in the hilus of ShhCre/+;Ai14 mice 1 week after seizure induction. (E) In situ RNAscope detection of Shh mRNA (green) in the DG from anterior to posterior 24 hours after seizure induction. (F) Representative RNAscope-immunofluorescence images for Shh expression in mossy cells labeled with GluR2/3 (red). (G) Quantification of Shh mRNA puncta in GluR2/3+ mossy cells. Values represent mean ± SEM; ****P < 0.0001. Unpaired t-test (two-tailed). A total of 608 and 584 GluR2/3+ cells were quantified from three mice in naive and KA treated groups, respectively.

Deletion of Shh in mossy cells decreases Shh signaling activation and attenuates seizure-induced neurogenesis.

(A) Representative Gli1-LacZ staining images of the DG of control (Crlr-Cre Shh+/+ and Shh+/+, Shh fl/+ and Shh fl/fl without Cre), and Crlr-Cre;Shhfl/fl (Shh-cKO);Gli1nlacZ/+ mice 3 days after KA-induced seizure. (B) Quantification of Gli1-nLacZ+ cells in SGZ. Values represent mean ± SEM; ns: P > 0.05, *P < 0.05, **P < 0.01, ****P < 0.0001. Two-way ANOVA with Tukey’s multiple comparison test. (C) Experimental scheme of analyzing neurogenesis by BrdU pulse labeling. One day after KA-induced seizures, control and Shh-cKO mice were injected with BrdU for 5 days and analyzed 3 days after the last BrdU injection. (D) Representative immunofluorescence images for newborn neurons labeled with BrdU (green), DCX (red) and DAPI (blue) in the SGZ of control and Shh-cKO mice after seizure induction (KA). (E) Quantification of newborn neurons after seizure induction in the SGZ of control and Shh-cKO mice. Values represent mean ± SEM; ns: P > 0.05, ****P < 0.0001. Two-way ANOVA with Tukey’s multiple comparison test.

Mossy cell neuronal activity induces neurogenesis though Shh.

(A) Schematic of conditional expression of DREADD activator in mossy cells. (B and C) Representative immunofluorescence images of DREADD activator hM3Dq/mCherry (red) expressing cells in the hippocampus (B) and in GluR2/3+ (green) mossy cells of dentate hilus (C). (D) Quantification of recombination specificity (% of GluR2/3+ mossy cells in mCherry+ recombined cells) and rate (% of mCherry+ recombined cells in GluR2/3+ mossy cells) in GluR2/3+ mossy cells. Values represent mean ± SEM. (E) Representative immunofluorescence images of neuronal activation (c-fos, green) in the hilus of RC::L-hM3Dq and Crlr-Cre;hM3Dq mice at 1.5 h after CLZ injection. hM3Dq expressing recombined cells are labeled with mCherry (red). (F) Quantification of c-fos+ cells in the hilus of RC::L-hM3Dq and Crlr-Cre;hM3Dq mice 1.5 hours after CLZ injection. Values represent mean ± SEM; *P < 0.05. Unpaired t-test (two-tailed). (G) Induction rate of neuronal activation in the hM3Dq expressing recombined cells (% of c-fos+ cells in mCherry+ cells in the hilus). Values represent mean ± SEM. (H) Experimental scheme of analyzing neurogenesis by BrdU pulse labeling during DREADD activation. CLZ was administered to two-month-old (2m) mice for 6 days. Starting on the 2nd day of CLZ administration, mice concomitantly received BrdU for 5 days and were analyzed 3 days after the last BrdU injection. (I) Representative immunofluorescence images for DCX+ (green) BrdU+ (red) newborn neurons produced after DREADD induced neuronal activation of mossy cells in the SGZ of RC::L-hM3Dq and Crlr-Cre;hM3Dq mice (J) Quantification of newborn neurons in the SGZ. Values represent mean ± SEM; **P < 0.01. Unpaired t-test (two-tailed). (K) Experimental scheme of analyzing Shh signaling activation after induction of mossy cell neuronal activity. (L) Representative Gli1-nLacZ staining images of the DG of RC::L-hM3Dq and Crlr-Cre;hM3Dq mice after CLZ administration for 6 days. (M) Quantification of Gli1-nLacZ+ cells in SGZ. Values represent mean ± SEM; *P < 0.05. Unpaired t-test (two-tailed). (N) Representative immunofluorescence images for newborn neurons produced after DREADD induced neuronal activation of mossy cells in the SGZ of Shhfl/fl;RC::L-hM3Dq and Crlr-Cre; Shhfl/fl;hM3Dq mice, which are labeled with DCX (green), BrdU (red) and DAPI (blue). (O) Quantification of newborn neurons in the SGZ of Shhfl/fl;RC::L-hM3Dq and Crlr-Cre; Shhfl/fl;hM3Dq mice after DREADD induced neuronal activation of mossy cells. Values represent mean ± SEM; ns: P > 0.05. Unpaired t-test (two-tailed). (P) Our proposed model of mossy cell mediated neurogenesis after seizures. Shh expression in mossy cells are upregulated by seizure activity. Neuronal activity of mossy cells activates Shh signaling in NSCs and contributes to the seizure-induced neurogenesis.

Shh from mossy cells maintains NSC pool after seizure induced neurogenesis.

(A) Experimental scheme of long-term tracing of NSCs that proliferate after seizure induction. Seizures were induced in two-month-old (2m) control and Shh-cKO mice by KA injections. One day after seizure induction, mice received BrdU via drinking water for 5 days and were analyzed on the last day of BrdU administration (day 0) and at 4 weeks after BrdU administration. (B) Schematic of NSC fate after proliferation. (C) Representative immunofluorescence images of proliferating Sox2+ (green) NSCs labeled with BrdU (red) on day 0 and radial GFAP+ (cyan) Sox2+ (green) NSCs retaining BrdU (red) at 4 weeks after BrdU administration, which are indicated by white arrowheads. (D) Quantification of Sox2+ BrdU+ proliferating NSCs after seizure induction on day 0. Values represent mean ± SEM; ns: P > 0.05, **P < 0.01, ****P < 0.0001. Two-way ANOVA with Tukey’s multiple comparison test. (E) Quantification of Sox2+ GFAP+ radial NSCs retaining BrdU at 4 weeks after BrdU administration. Values represent mean ± SEM; ns: P > 0.05, ****P < 0.0001. Two-way ANOVA with Tukey’s multiple comparison test. (F) Quantification of NSC persistence 4 weeks after BrdU administration in naive and KA-induced mice. Values represent mean ± SEM; ns: P > 0.05, **P < 0.01. Two-way ANOVA with Tukey’s multiple comparison test. (G) Representative immunofluorescence images of Sox2+ (green) GFAP+ (red) radial NSCs at 4 weeks after BrdU administration (5 weeks after KA-induced seizure). (H) Quantification of Sox2+ GFAP+ radial NSCs at 5 weeks after seizure induction in control and Shh-cKO mice. Values represent mean ± SEM; ns: P > 0.05, **P < 0.01. Two-way ANOVA with Tukey’s multiple comparison test.

deletion of Shh in mossy cells increases age-related NSC decline.

(A) Representative immunofluorescence images of Sox2+ (green) GFAP+ (red) radial NSCs in 9- to 11-month-old control and Shh-cKO mice. High magnification of inset images are shown in the right panels. (B) Quantification of Sox2+ GFAP+ radial NSCs. Values represent mean ± SEM; *P < 0.05. Unpaired t-test (two-tailed). (C) Experimental scheme of long-term tracing of proliferating NSCs in aged mice. 9- to 11-month-old control and Shh-cKO mice were administrated with BrdU via drinking water for 5 days and were analyzed on the last day of BrdU administration (day 0), and at 10 days and 4 weeks after BrdU administration. Schematic of NSC fate after proliferation. (D) Representative immunofluorescence images of proliferating Sox2+ (green) NSCs labeled with BrdU (cyan) at day 0 and radial Sox2+ (green) GFAP+ (red) NSCs retaining BrdU (cyan) at 10 days and 4 weeks after BrdU administration. (E) Quantification of the fraction of NSCs retaining BrdU until 10 days and 4 weeks after BrdU administration in aged control and Shh-cKO mice. Values represent mean ± SEM; *P < 0.05, **P < 0.01. Unpaired t-test (two-tailed).

Shh-responding cells in the SGZ are NSCs.

(A) Experimental scheme of tamoxifen injection to label Gli1 expressing cells in Gli1CreER/+;Ai14 mice. (B) Representative immunofluorescence images of Gli1 expressing cells labeled with tdTomato (red) in the DG one day after a three-day tamoxifen treatment. NSCs are defined as stem cell marker Sox2+ (cyan) positive and astrocyte marker S100B (green) negative cells in upper panels (white arrowhead). Radial NSCs are labeled with Sox2 (green) and defined by radial fiber stained with GFAP (cyan) in bottom panels (yellow arrowheads). (C) Quantification of Sox2+ S100B-NSCs in tdTomato+ cells and Sox2+ GFAP+ radial NSCs in tdTomato+ Sox2+ cells. Values represent mean ± SEM.

Shh is expressed in dorsal mossy cells.

(A) Schematic illustration of coronal sections of hippocampus (reference images of the coronal mouse brain from Allen Brain Atlas). (B) Representative immunofluorescence images of DG from ShhCre/+;Ai14 mice 1 week after seizure induction. Higher magnification of inset panels from dorsal and ventral regions of posterior DG. GluR2/3+ (green) mossy cells in dorsal but not ventral DG were labeled with ShhCre/+;Ai14 (red).

Deletion of Shh from mossy cells does not change NSC number.

(A) Representative immunofluorescence images of Sox2+ (green) GFAP+ (red) radial NSCs in the SGZ of DG in control and Shh-cKO mice. Higher magnification of inset panels representing radial NSCs labeled with Sox2 and GFAP. (B) Quantification of Sox2+ GFAP+ radial NSCs in the SGZ. Values represent mean ± SEM; ns: P > 0.05. Unpaired t-test (two-tailed)

Crlr-Cre mediated recombination in the DG.

Representative immunofluorescence images of Crlr-Cre recombination in cells labeled with mCherry (red) in the anterior to posterior DG of Crlr-Cre;hM3Dq mice.

Neuronal activity of mossy cells induces neurogenesis in the contralateral DG.

(A) Schematic of AAV injection into the dorsal DG of Crlr-Cre mice. Either AAV::hSyn-DIO-mCherry (as control) or AAV::hSyn-DIO-hM3Dq-mCherry was injected into the dorsal DG of Crlr-Cre mice. (B) Representative immunofluorescence images of AAV::mCherry infection (red) in the DG of Crlr-Cre mice ipsilateral and contralateral to the AAV injection site. Projections of mossy cells were observed in the molecular layer (ML) in the contralateral DG. (C) Representative immunofluorescence images of AAV::hM3Dq-mCherry infection (red) in GluR2/3+ mossy cells (green) in the AAV injected DG of Crlr-Cre mice. (D) Quantification of AAV::hM3Dq-mCherry infection specificity (% of GluR2/3+ mossy cells in mCherry+ infected cells) and fraction (% of mCherry+ infected cells in GluR2/3+ mossy cells) in GluR2/3+ mossy cells in the AAV injected side of the entire (total) and dorsal DG of Crlr-Cre mice. Values represent mean ± SEM. (E) Representative immunofluorescence images of neuronal activation in the hilus of AAV::hM3Dq-mCherry injected Crlr-Cre mice 1.5 hours after CLZ injection. (F) Quantification of c-fos+ cells in the hilus of AAV::hM3Dq-mCherry injected Crlr-Cre mice 1.5 hours after CLZ injection. Values represent mean ± SEM; ****P < 0.0001. Unpaired t-test (two-tailed). (G) Induction rate of neuronal activation in the hM3Dq expressing infected cells (% of c-fos+ cells in mCherry+ cells in the hilus) in the DG ipsilateral to AAV injections in AAV::hM3Dq-mCherry injected Crlr-Cre mice. Values represent mean ± SEM. (H) Experimental scheme for AAV injection and analysis of neurogenesis by BrdU pulse labeling in DREADD mediated neuronal activation. AAV was injected into 1 month old Crlr-Cre mice. After 3-4 weeks recovery, mice were administered with CLZ for 6 days. Starting on the second day of CLZ administration, mice concomitantly received BrdU for 5 days and were analyzed 3 days after last BrdU injection, neurogenesis in the contralateral DG was analyzed. (I) Representative immunofluorescence images for DCX+ (green) BrdU+ (red) newborn neurons produced after DREADD-induced neuronal activation of mossy cells in the SGZ of the contralateral DG. (J) Quantification of newborn neurons in the SGZ of contralateral DG in AAV injected Crlr-Cre mice. Values represent mean ± SEM; **P < 0.01. Unpaired t-test (two-tailed).

Deletion of Shh attenuates seizure induced neurogenesis in the contralateral DG.

(A) Schematic of AAV injection into the dorsal DG of Crlr-Cre mice. Either AAV::hSyn-mCherry (AAV::mCherry, as control) or AAV::hSyn-Cre-P2A-tdTomato (AAV::Cre-tdTomato) was injected into the dorsal DG of Shhfl/fl mice. (B) Representative immunofluorescence images of AAV::Cre-tdTomato infection (red) in the AAV injected side and contralateral DG. AAV-infection was observed in the hilus of dorsal DG. Contralateral projection of mossy cells was observed in the molecular layer (ML) of contralateral DG. (C) Representative immunofluorescence images of GluR2/3+ (green) mossy cells infected with AAV::Cre-tdTomato (red) in the AAV injected side and contralateral DG. (D) Quantification of infection in GluR2/3+ mossy cells (% of mCherry+ infected cells in GluR2/3+ mossy cells) in the entire (total) and dorsal DG of AAV-injected side. Values represent mean ± SEM. (E) Experimental scheme of AAV injection and analyzing neurogenesis by BrdU pulse labeling after seizure induction by KA. AAV was injected into 1 month old Shhfl/fl mice. After 3-4 weeks recovery, seizure was induced by KA administration. One day after seizure induction, mice were given BrdU for 5 days and analyzed 3 days after last BrdU injection. Neurogenesis in the contralateral DG was analyzed. (F) Representative immunofluorescence images for DCX+ (red) BrdU+ (green) newborn neurons produced after seizure in the SGZ of the contralateral DG in AAV-injected Shhfl/fl mice. (G) Quantification of newborn neurons in the SGZ of the contralateral DG in AAV-injected Shhfl/fl mice. Values represent mean ± SEM; ns: P > 0.05, *P < 0.05, ****P < 0.0001. Two-way ANOVA with Tukey’s multiple comparison test.

Expressing DREADD activator does not affect neurogenesis and number of NSCs of Crlr-Cre Shh mice.

(A) Experimental scheme of analyzing neurogenesis by BrdU pulse labeling. The mice were injected with BrdU for 5 days and analyzed 3 days after last BrdU injection. (B) Representative immunofluorescence images for newborn neurons labeled with BrdU (green), DCX (red) and DAPI (blue) in the SGZ of control (Cre negative mice), Crlr-Cre Shhfl/fl, Shhfl/fl;RC::L-hM3Dq and Crlr-Cre Shhfl/fl; RC::L-hM3Dq mice without CLZ administration. (C) Quantification of newborn neurons in the SGZ. Values represent mean ± SEM; ns: P > 0.05. One-way ANOVA with Tukey’s multiple comparison test. (D) Representative immunofluorescence images of radial NSCs labeled with Sox2 (green), GFAP (red) and DAPI (blue) in the SGZ of two-month-old mice. (E) Quantification of Sox2+ GFAP+ radial NSCs in the SGZ. Values represent mean ± SEM; ns: P > 0.05. One-way ANOVA with Tukey’s multiple comparison test. (F) Representative immunofluorescence images of radial NSCs labeled with Sox2 (green), GFAP (red) and DAPI (blue) in the SGZ of 9-to 11-month-old mice. (G) Quantification of number of GFAP+ Sox2+ radial NSCs in the SGZ. Values represent mean ± SEM; ns: P > 0.05, *P < 0.05, **P<0.01. One-way ANOVA with Tukey’s multiple comparison test.