Effects of long‑term scopolamine treatment on cognitive deficits and calcium binding proteins immunoreactivities in the mouse hippocampus

Abstract

GABAergic projections terminate on numerous hippocampal interneurons containing calcium binding proteins (CBPs), including calbindin D‑28k (CB), calretinin (CR) and parvalbumin (PV). Memory deficits and expression levels of CB, CR, and PV were examined in the hippocampal subregions following systemic scopolamine (Scop; 1 mg/kg) treatment for 4 weeks in mice. Scop treatment induced significant memory deficits from 1 week after Scop treatment. CB, CR and PV immunoreactivities distributions were in hippocampal subregions [CA1 and CA3 regions, and the dentate gyrus (DG)]. CB immunoreactivity (CB+) was gradually decreased in all subregions until 2 weeks after Scop treatment, and CB+ was decreased to the lowest level in all subregions at 3 and 4 weeks. CR+ in the CA1 region was gradually decreased until 2 weeks and hardly observed at 3 and 4 weeks; in the CA3 region, CR+ was not altered in all subregions at any time. In the DG, CR+ was gradually decreased until 2 weeks and lowest at 3 and 4 weeks. PV+ in the CA1 region was not altered at 1 week, and gradually decreased from 2 weeks. In the CA3 region, PV+ did not change in any subregions at any time. In the DG, PV+ was not altered at 1 week, decreased at 2 weeks, and lowest at 3 and 4 weeks. In brief, Scop significantly decreased CBPs expressions in the hippocampus ≥3 weeks after the treatment although memory deficits had developed at 1 week. Therefore, it is suggested that Scop (1 mg/kg) must be systemically treated for ≥3 weeks to investigate changes in expression levels of CBPs in the hippocampus.

Figure 1.

Memory test using the MWMT (Aa-Ac) and PAT (B) in the control and Scop groups. In the MMWT, escape latency in all the Scop groups is significantly increased compared with that in the control group (Aa and Ab), and duration of the time in the target quadrant in all the Scop groups is significantly decreased compared with that in the control group (Ac). In the PAT, latency in all the Scop groups is significantly decreased compared with that in the control group (B). (n=7 per group; *P<0.05 vs. the control group). The bars indicate the SEM. MWMT, Morris water maze test; PAT, passive avoidance test; Scop, scopolamine.

Figure 2.

CB immunohistochemistry in the hippocampal subregions of the control (Aa-Ac), 1 wk-(Ba-Bc), 2 wk-(Ca-Cc), 3 wk-(Da-Dc), and 4 wk-(Ea-Ec) Scop groups. CB immunoreactivity in the control group is shown in cells the stratum pyramidale (SP, arrows) in the CA1 region, in the mossy fibers (arrows) in the CA3 region, and cells in the granule cell layer (GCL, arrow) in the DG. CB immunoreactivity in the Scop groups is gradually and significantly decreased with time in all the subregions. Scar bar, 50 µm. (F) ROD of CB immunoreactive structures in the control and Scop groups (n=7 per group; *P<0.05 vs. the control group; ^†P<0.05 vs. the pre-time point Scop group). The bars indicate the SEM. CB, calbindin D-28k; wk, week; Scop, scopolamine; SP, stratum pyramidale; GCL, granule cell layer; DG, dentate gyrus; ML, molecular layer; PL, polymorphic layer; SO, stratum oriens; SR, stratum radiatum; ROD, relative optical density.

Figure 3.

CR immunohistochemistry in the hippocampal subregions of the control (Aa-Ac), 1 wk-(Ba-Bc), 2 wk-(Ca-Cc), 3 wk-(Da-Dc), and 4 wk-(Ea-Ec) Scop groups. CR immunoreactivity in the control group is shown cells (arrows) scattered in the CA1 and CA3 region, and cells in the granule cell layer (GCL, arrow) and cells in the polymorphic layer (PL, arrow) in the DG. CR immunoreactivity is significantly reduced in the CA1 region and the DG with time after Scop treatment, but not in the CA3 region. Scar bar, 50 µm. (F) ROD of CR immunoreactive structures in the control and Scop groups (n=7 per group; *P<0.05 vs. the control group; ^†P<0.05 vs. the pre-time point Scop group). The bars indicate the SEM. CR, calretinin; wk, week; Scop, scopolamine; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; GCL, granule cell layer; PL, polymorphic layer; ML, molecular layer; DG, dentate gyrus; ROD, relative optical density.

Figure 4.

PV immunohistochemistry in the hippocampal subregions of the control (Aa-Ac), 1 wk-(Ba-Bc), 2 wk-(Ca-Cc), 3 wk-(Da-Dc), and 4 wk-(Ea-Ec) Scop groups. In the control group, strong PV immunoreactivity is shown in scattered cells (arrows) in the CA1, CA3 region, and DG, and weak PV immunoreactivity is detected in cells in the SP of the CA1 and CA3 regions, and in the GCL of the DG. PV immunoreactivity is gradually and significantly decreased in the CA1 region and the DG, not in the CA3 region, from 2 weeks after Scop treatment. Scar bar, 50 µm. (F) ROD of PV immunoreactive structures in the control and Scop groups (n=7 per group; *P<0.05 vs. the control group; ^†P<0.05 vs. the pre-time point Scop group). The bars indicate the SEM. PV, parvalbumin; wk, week; Scop, scopolamine; DG, dentate gyrus; GCL, granule cell layer; ML, molecular layer; PL, polymorphic layer; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; ROD, relative optical density.