Article Figures & Data
Figures
- Figure 1.
Tamalin mRNA expression is upregulated in the DG of adult mice after kainate administration and ECS. Representative in situ hybridization (a–d, g, h) and immunohistochemistry (e, f, i, j) analysis performed with tamalin (a, b, g, h) or homer1a (c, d) specific probes and an arc-specific antibody (e, f, i, j), respectively. Adult animals were injected with vehicle (a, c, e) or kainate (10 mg/kg, i.p.; b, d, f) and killed 3 h later for in situ hybridization or immunohistochemistry analysis. Homer1a and arc, established immediate early genes, were used as positive controls. Note that tamalin mRNA expression is upregulated in response to kainate (b) similarly to homer1a mRNA (d) and arc protein (f). ECS also causes an increase in Arc immunoreactivity (j) and tamalin mRNA (h) 3 h after treatment in the DG of the hippocampus when compared with control animals (i and g, respectively). n = 4 animals for each analysis.
- Figure 2.
Hippocampal tamalin mRNA and protein are upregulated in response to ECS. Representative tamalin in situ hybridization analysis of hippocampus from mice after single (a–f) or chronic (g, h) ECS. Sham-treated animals show the basal level of tamalin expression (a, g). Tamalin mRNA is upregulated at 1 h (b) and peaks at 3 h (c) from ECS. Beginning at 6 h (d), tamalin is downregulated and returns to the pre-ECS level by 12 (e) and 24 h (f). Note that tamalin mRNA expression remains upregulated at 24 h after chronic ECS (h) compared with sham-treated controls (g). n = 4 for each group. Expression of tamalin protein after chronic ECS (i). Animals received one ECS per day for 5 consecutive days and were killed 24 h after the last treatment. Western blot analysis of hippocampus (left), olfactory bulb (middle), and cortex (right) from ECS-treated (E) or sham-treated (C) WT animals using an anti-tamalin antibody (Tam); β-actin was used as loading control (Act), and a hippocampus lysate from a TKO mouse was used as negative control (TKO). The quantification of tamalin protein levels is shown at the bottom. Note the selective tamalin upregulation in the hippocampus. n = 4 animal. p < 0.05 by Student's t test.
- Figure 3.
Targeted deletion of tamalin in mouse. Schematic representation of the strategy used to target the tamalin gene. LoxP sites (black triangles) were inserted upstream and downstream of the ATG-containing exon 1 (EX1). The neomycin resistance cassette (neo) is flanked by both loxP and Frt (black rectangles) sites. After the initial screening of the positive ES cell clones, as described in Material and Methods, a probe (black rectangle) downstream of EX1 was used for the identification of the different targeted alleles by detecting a shift from the endogenous BamHI WT band of 8 kb to the rearranged 9.8 kb band resulting from the insertion of the neo cassette by homologous recombination. Cre-induced excise of the tamalin-specific exon and the neo cassette causes a shift of the 9.8 kb band to 7.3 kb. b, Southern blot analysis of genomic DNA from mice with the tamalin alleles indicated in a. Analysis of DNA digested with the BamHI restriction enzyme and probed with the 3′ fragment described in a yields the expected 8 kb band in a WT mouse (lanes 1) and a rearranged 9.8 kb fragment with the neo insertion (floxneo) in an heterozygous mouse and a 7.3 kb band in the mutant (lanes 2 and 3, respectively). c, Western blot analysis of hippocampus dissected from mutant (KO) or WT mice and hybridized with an antiserum directed against tamalin. The top panel is a Western blot analysis of lysates subjected first to immunoprecipitation (IP) with a tamalin-specific antibody and then to the blotting with the same antibody. The bottom panel is the Western blot analysis of straight hippocampus lysates hybridized with the tamalin-specific antibody. The middle blot was hybridized with an anti-β-actin-specific antibody to control for loading. Behavioral analysis of tamalin-deficient mice (d–f). Animals were subjected to the elevated plus maze (d), forced swim (e), and rotarod (f) test. n ≥ 7.
- Figure 4.
Tamalin deficiency blocks ECS-induced proliferation of adult hippocampal progenitors. BrdU staining of representative hippocampal sections from WT (a) and tamalin KO (b) mice showing BrdU+ hippocampal progenitors in the subgranular zone (SGZ, arrowhead) of the DG, 2 h after BrdU injection. c, d, Quantification of BrdU+ cells in the subgranular zone of the hippocampus showing no changes in basal progenitors proliferation (c) or survival (d) in WT and TKO mice. The data analyzed by Student's t test represent the mean ± SEM; n = 4–5 per group. e–h, Representative BrdU immunohistochemistry images from sham-treated (e, f) or ECS-treated (g, h) WT (e, g) and TKO (f, h) animals. Note the mild, nonsignificant, increase of ECS-induced BrdU+ proliferating progenitors in the subgranular zone of the DG of TKO mice compared with the robust increase in the WT animals (i). j–m, Representative photomicrographs of PCNA+ cells as in e–h. Note the significant increase in the number of PCNA+ cells only in the ECS-treated WT animals (n). Data in i and n was analyzed by two-way ANOVA, followed by post hoc Bonferroni's test and represent the mean ± SEM. n = 8–10 per group. Two-way ANOVA revealed a significant ECS and genotype interaction for data presented in i (F(1,14) = 6.53, p < 0.05) and n (F(1, 32) = 5.87, p < 0.05).The schedules of BrdU injection and analysis for the basal proliferation (c), survival (d), and ECS-induced proliferation (i, n) are depicted at the top. GCL, Granular cell layer.
- Figure 5.
Tamalin is required for ECS-induced hippocampal neurogenesis. a–d, Representative immunohistochemistry images showing DCX+ (a marker for immature neurons) cells in the DG of WT (a, c) and TKO (b, d) animals subjected to sham (a, b) or ECS (c, d) treatment. e, Quantitative analysis of DCX+ cells showing an increase in the number of immature neurons caused by ECS in WT but not in TKO animals. n = 6–8 animals per group. f–h, Confocal z-stack representative images showing colocalization of BrdU+ cells with NeuN, a marker for mature neurons. WT and TKO animals were subjected to a single sham or ECS treatment, and they were injected with BrdU (200 mg/kg, i.p.) 3 d later. i, Quantitative analysis showing the increase in the number of BrdU/NeuN double-positive cells caused by ECS in WT animals but not in TKO mice. n = 4–7 animals per group. Data represent the mean ± SEM analyzed by two-way ANOVA, followed by Bonferroni's post hoc test. Two-way ANOVA demonstrated a significant ECS × genotype interaction in both the DCX (e) (F(1, 24) = 8.01, p < 0.05) and NeuN/BrdU (i) (F(1,19) = 8.77, p < 0.05) analysis. The schedules of BrdU injection and DCX or BrdU/NeuN analysis are depicted at the top.
- Figure 6.
Chronic ECS-induced neuronal sprouting is blunted in TKO mice. Analysis of mossy fiber sprouting by Timm staining (a–e) and dendritic complexity of dentate granule cells by Golgi–Cox staining (f–j). Representative Timm-stained hippocampal sections from sham-treated (a, b) or chronic ECS-treated (c, d) WT (a, c) or TKO (b, d) mice. Note the reduced presence of Timm granules (white arrowheads in c, d) in the granular cell layer (GCL) of the hippocampus of ECS-treated TKO mice compared with ECS-treated control mice (n ≥ 5 animals for each group). e, Quantitative analysis of Timm scores. Data representing the mean ± SEM was analyzed by nonparametric ANOVA, followed by post hoc Dunn test. f, Representative Neurolucida reconstruction of DG Golgi-stained neurons from sham-treated or chronic ECS-treated WT and TKO mice. g–j, Sholl analysis of dendritic intersection number and dendrite length of DG neurons showing no difference between sham-treated WT and TKO mice (g–i; n = 5 mice in each group; 15 neurons per mouse). Only ECS-treated WT animals display a significant increase in the number of intersections (h) and dendritic length (j) compared with sham-treated controls (n ≥ 9 mice in each group; 15 neurons per mouse). *p < 0.05. Data represent the mean ± SEM analyzed by two-way ANOVA, followed by Bonferroni's post hoc test. Top, Schematic indicating that sham-treated or chronic ECS-treated (once per day for 10 consecutive days) animals were killed 12 d after the last ECS. IML, Inner molecular layer.
- Figure 7.
Tamalin deficiency blocks LTP formation after ECS. Three 100 Hz, 1-s-long stimuli trains, induced an immediate posttetanic potentiation lasting a few minutes then a long-term potentiation lasting several hours (LTP). a, Time course of fEPSPs evoked in the molecular layer of the DG by stimulating the medial perforant path. Slope at baseline (−10 to 0 min) and after induction of LTP (0 time mark). Points are average of traces from four WT and four TKO with and without ECS. For animals without ECS, no differences are evident in TKO with respect to WT mice. ECS blunted the LTP formation in TKO but did not alter posttetanic potentiation. Note that ECS administered 5 h before LTP recording blunted the response in WT but the reduction in LTP was much more pronounced in the TKO mice. b, fEPSP recorded in DG. Traces from top to bottom identify baseline fEPSP before LTP induction, 1 min after induction, and 1 h after induction. c, Input–output curve obtained before LTP induction in TKO and WT mice that underwent ECS.
