Recruitment of the Sonic hedgehog signalling cascade in electroconvulsive seizure-mediated regulation of adult rat hippocampal neurogenesis

Abstract

Electroconvulsive seizure (ECS) induces structural remodelling in the adult mammalian brain, including an increase in adult hippocampal neurogenesis. The molecular mechanisms that underlie this increase in the proliferation of adult hippocampal progenitors are at present not well understood. We hypothesized that ECS may recruit the Sonic hedgehog (Shh) pathway to mediate its effects on adult hippocampal neurogenesis, as Shh is known to enhance the proliferation of neuronal progenitors and is expressed in the adult basal forebrain, a region that sends robust projections to the hippocampus. Here we demonstrate that the ECS-induced increase in proliferation of adult hippocampal progenitors was completely blocked in animals treated with cyclopamine, a pharmacological inhibitor of Shh signalling. Our results suggest that both acute and chronic ECS enhance Shh signalling in the adult hippocampus, as we observed a robust upregulation of Patched (Ptc) mRNA, a component of the Shh receptor complex and a downstream transcriptional target of Shh signalling. This increase was rapid and restricted to the dentate gyrus, where the adult hippocampal progenitors reside. In addition, both acute and chronic ECS decreased Smoothened (Smo) mRNA, the other component of the Shh receptor complex, selectively within the dentate gyrus. However, ECS did not appear to influence Shh expression within the basal forebrain, the site from which it has been suggested to be anterogradely transported to the hippocampus. Together, our findings demonstrate that ECS regulates the Shh signalling cascade and indicate that the Shh pathway may be an important mechanism through which ECS enhances adult hippocampal neurogenesis.

Fig. 1

Cyclopamine prevents the electroconvulsive seizure-mediated increase in the proliferation of dentate granule cell progenitors. Rats received i.c.v. administration of either vehicle or cyclopamine through osmotic minipumps prior to the administration of sham or electroconvulsive seizure (ECS) treatment, as described in Materials and methods. The number of proliferating progenitors within the subgranular zone (SGZ)/granule cell layer (GCL) of the dentate gyrus was determined using the mitotic marker BrdU. ECS administration resulted in a significant increase in the number of BrdU-positive cells per section within the dentate gyrus of the vehicle + ECS group (B; *P < 0.001 as compared with the Vehicle + Sham treatment). Cyclopamine pretreatment significantly reduced the number of BrdU-positive cells per section in the Cyclopamine + Sham group (B; *P < 0.001 as compared with the Vehicle + Sham treatment group). ECS administration to animals pretreated with cyclopamine (Cyclopamine + ECS group; B) did not significantly increase the number of BrdU-positive cells within the dentate gyrus as compared with the Cyclopamine + Sham treatment, indicating that cyclopamine prevents the ability of ECS to enhance the proliferation of dentate granule cell progenitors. The Cyclopamine + ECS group had significantly decreased numbers of BrdU-positive cells per section as compared with the Vehicle + ECS group (δP < 0.001; B). Shown are representative images of the dentate gyrus region from Vehicle + Sham, Vehicle + ECS, Cyclopamine + Sham and Cyclopamine + ECS groups (A). BrdU-positive cells (arrow A) were observed in the SGZ, at the border of the hilus (H) and the GCL. The results are expressed as the mean ± SEM (n = 5 per group) number of BrdU-positive cells per section in the dentate gyrus. *P < 0.001 indicates significantly different from Vehicle + Sham; δP < 0.001 indicates significantly different from Vehicle + ECS (ANOVA and Bonferroni post-hoc test).

Fig. 2

Influence of acute and chronic electroconvulsive seizure (ECS) administration on the expression of Sonic hedgehog (Shh) mRNA within the vertical limb of the diagonal band (VDB), and Shh protein within the VDB and hippocampus. Rats received either acute or chronic ECS/sham treatment as described in Materials and methods, and the levels of Shh mRNA in the VDB were determined using in situ hybridization analysis and quantitative densitometry. Representative autoradiograms from a sham and acute ECS-treated animal are shown with an arrow indicating the VDB (A). Levels of Shh mRNA in the VDB were not altered by either acute or chronic ECS treatment (B). The results are expressed as the mean ± SEM percentage of sham (Acute ECS experiment n = 9 per group; Chronic ECS experiment, n = 8 per group; Student’s t-test). Shh immunoreactivity was observed in cell bodies within the VDB, and neither the number of immunopositive cells nor the intensity of staining appeared to be altered following either acute or chronic ECS treatment (C; n = 4 per group). Shown are representative fluorescence photomicrographs of Shh immunoreactivity in the VDB from a sham and acute ECS-treated animal (C). Representative immunoblots (D) show Shh levels in the VDB and hippocampus after sham (S) or chronic ECS (E) treatment, Shh represents the positive control of purified N-terminal Shh protein. Neither acute nor chronic ECS was found to alter the expression of Shh protein levels in the VDB or the hippocampus. Levels of Shh immunoreactivity following sham or chronic ECS treatment are represented as arbitrary OD units and results are expressed as mean + SEM (n = 4 per group, P < 0.05, Student’s t-test).

Fig. 3

Acute and chronic electroconvulsive seizures (ECS) regulate the expression of the Shh receptor components Patched (Ptc) and Smoothened (Smo) in the dentate gyrus. Rats received either acute or chronic sham/ECS treatment as described in Materials and methods, and mRNA levels of the Shh receptors, Ptc and Smo in the hippocampal dentate gyrus subfield were determined using in situ hybridization analysis and quantitative densitometry. Representative autoradiographs of Ptc (A) and Smo (B) mRNA expression from a sham and acute ECS-treated animal are shown with an arrow indicating the dentate gyrus (DG). Levels of Ptc mRNA and Smo mRNA in the dentate gyrus hippocampal subfield are shown in the bar graphs below the representative autoradiograms. Acute and chronic ECS significantly enhanced the expression of Ptc mRNA within the dentate gyrus, whereas the expression of Smo mRNA was significantly decreased following both acute and chronic ECS treatment. The results are expressed as the mean ± SEM percentage of sham (Acute ECS experiment n = 9 per group; Chronic ECS experiment, n = 8 per group). *P < 0.05 indicates significantly different from sham (Student’s t-test). Cellular localization of the ECS induction of Ptc mRNA and the downregulation of Smo mRNA was visualized with emulsion autoradiography and then counterstained with Cresyl violet. Shown are representative emulsion sections through the granule cell layer (GCL) for Ptc (C) and Smo (D) mRNA from a sham and acute ECS-treated animal. Note the presence of black silver grains, in both the Ptc and Smo emulsion, through the entire GCL including the subgranular zone (SGZ) at the border of the GCL and the hilus. Shown are representative images indicating the expression of Ptc mRNA (purple, E) and Smo mRNA (purple, F) in doublecortin-immunopositive adult hippocampal progenitors (arrowhead) within the SGZ.

Fig. 4

Influence of pretreatment with the NMDA receptor antagonist 5-methyl-10,11-dihydro-5H-dibenzo (a and d) cyclohepten-5,10-imine maleate (MK-801) on the acute ECS-mediated regulation of Patched (Ptc) and Smoothened (Smo) mRNA. MK-801 (3 mg/kg) was administered 30 min prior to sham/acute ECS treatment as described in Materials and methods. The levels of Ptc and Smo mRNA within the dentate gyrus (DG) were determined using in situ hybridization analysis and quantitative densitometry. Representative autoradiograms for Ptc (A) and Smo (B) mRNA expression in the dentate gyrus from the Vehicle + Sham, Vehicle + Acute ECS, MK-801 + Sham and MK-801 + Acute ECS groups are shown. Levels of Ptc mRNA and Smo mRNA in the dentate gyrus hippocampal subfield are shown in the bar graphs below the representative autoradiograms. Acute ECS significantly increased the expression of Ptc mRNA and decreased Smo mRNA within the dentate gyrus (*P < 0.05 as compared with Vehicle + Sham). Pretreatment with MK-801 did not significantly influence either the basal expression or the ECS-mediated increase in Ptc mRNA levels (A). MK-801 treatment resulted in a significant increase in the basal expression of Smo mRNA levels, but did not influence the ECS-mediated decrease in Smo mRNA expression. The results are expressed as the mean ± SEM percentage of Vehicle + Sham (n = 4 per group). *P < 0.05 indicates significantly different from Vehicle + Sham; #P < 0.05 indicates significantly different from MK-801 + Sham (ANOVA and Bonferroni post-hoc test).

Fig. 5

Influence of pretreatment with the AMPA/KA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) on the acute electroconvulsive seizures (ECS)-mediated regulation of Patched (Ptc) and Smoothened (Smo) mRNA. DNQX (12.5 mg/kg) was administered 30 min before sham/acute ECS treatment, as described in Materials and methods. The mRNA levels of Ptc and Smo within the dentate gyrus were determined using in situ hybridization analysis and quantitative densitometry. Representative autoradiogram images of Ptc (A) and Smo (B) mRNA expression in the dentate gyrus from the following groups: Vehicle + Sham, Vehicle + Acute ECS, DNQX + Sham and DNQX + Acute ECS are shown. Levels of Ptc and Smo mRNA in the dentate gyrus hippocampal subfield are shown in bar graphs below the representative autoradiograms. Acute ECS significantly upregulated the expression of Ptc mRNA, and downregulated Smo mRNA within the dentate gyrus (*P < 0.05 as compared with Vehicle + Sham). Pretreatment with DNQX did not significantly influence either the basal expression or the ECS-mediated increase in Ptc mRNA levels (A). DNQX treatment resulted in a significant upregulation in the basal expression of Smo mRNA levels but did not influence the ECS-mediated downregulation of Smo mRNA expression. The results are expressed as the mean ± SEM percentage of Vehicle + Sham (n = 3 per group). *P < 0.05 indicates significantly different from Vehicle + Sham; #P < 0.05 indicates significantly different from MK-801 + Sham (ANOVA and Bonferroni post-hoc test).