Article Figures & Data
Figures
- Figure 1.
PTZ induces a single, self-limited, generalized seizure. Continuous vEEG recordings were performed in saline injected (Sham) and PTZ-induced animals for 24 h after induction. Depicted are representative hippocampal EEG traces from implanted rats before PTZ seizure induction (baseline), at the time of seizure (induction), and at time points used for behavioral training and testing. A, Baseline. No overt difference in baseline EEG activity was observed. B, Induction. Following saline administration (black arrow), no difference in EEG activity was observed in Sham animals (top trace). However, PTZ-seizure induction (black arrow) resulted in abnormal electrographic spikes followed immediately by a single, self-limited discharge of high-amplitude spikes observed in both the cortex (data not shown) and hippocampus (bottom trace). Red bars indicate seizure onset and termination in the hippocampus with similar findings in the cortex (data not shown). At the onset of the spike discharges (electrographic seizure onset), a behavioral Racine stage 4 generalized seizure was observed. C, Postinduction. Representative EEG traces for Sham (left) and PTZ-induced animals (right) at 1, 3, 16, and 24 h after induction. There were no other seizure events in PTZ-induced animals when compared with Sham controls at any time point during the 24 h after induction (n = 4–5/group).
- Figure 2.
A single generalized seizure does not significantly impair FC task acquisition. A, Timeline of seizure induction and the FC training protocol for short- as well as long-term memory. B, Training for short-term memory (left panel). When placed in a novel arena before the presentation of the CS-US (Pre CS-US), both Sham and PTZ animals displayed a low basal level of freezing which was not significantly different between the two groups. Following the presentation of the CS (Post CS-US), both Sham and PTZ animals had a significant increase in freezing levels (n = 6–8/group, **p < 0.01, ***p < 0.001). Training for long-term memory (right panel). Again, before CS-US presentation, both Sham and PTZ animals displayed a low basal level of freezing, which was not significantly different between the two groups. Both Sham and PTZ animals had a significant increase in freezing levels Post CS-US (n = 9/group, ***p < 0.001). There was no significant difference between Sham and PTZ animals Post CS-US. Data are presented as mean ± SEM.
- Figure 3.
Wort partially rescues memory deficits that result from a single PTZ-induced generalized seizure. A, Timeline detailing seizure induction, Wort administration, and the FC protocols. Two separate cohorts of animals were tested for short-term (cohort 1) and for long-term (cohort 2) memory. B, Contextual short-term memory. PTZ + Veh animals displayed significantly reduced freezing levels relative to Sham + Veh controls (n = 11–12/group). Wort treatment resulted in significantly increased freezing levels in PTZ + Wort animals relative to the PTZ + Veh condition (n = 11–12/group). There was no significant difference between Sham + Veh and PTZ + Wort animals (n = 11–12/group) and between the Sham + Wort group as compared with Sham + Veh animals (n = 11–12/group). C, Cued short-term memory. Before the CS presentation (left panel), there was no significant difference between the PTZ + Veh and Sham + Veh groups (n = 12–13/group). The PTZ + Wort animals displayed significantly elevated freezing levels when compared with the PTZ + Veh group (n = 12–13/group). There were no significant differences in freezing levels between the Sham + Veh and Sham + Wort conditions. During the CS presentation (right panel), the PTZ + Veh animals displayed significantly reduced freezing levels when compared with Sham + Veh controls (n = 11–12/group). While there remained a significant reduction in freezing levels in the PTZ + Wort animals compared with Sham + Veh controls (n = 11–12/group), PTZ + Wort animals displayed significantly increased freezing levels compared with PTZ + Veh animals (n = 12–13/group). There was no difference in freezing levels between Sham + Veh and Sham + Wort animals. D, Contextual long-term memory. As we observed for short-term memory, there was a significant reduction in the freezing levels in PTZ + Veh animals relative to the Sham + Veh control group (n = 11–15/group). There was no significant difference between PTZ + Veh and PTZ + Wort animals (n = 11–15/group). Wort did not significantly affect freezing levels in the Sham + Wort group relative to Sham + Veh controls (n = 11–15/group). E, Cued long-term memory. As compared with Sham + Veh controls, all other treatment groups displayed significantly reduced freezing levels in the Pre CS (left panel) condition (n = 11–15/group). No other significant differences were observed. During the presentation of the CS (right panel), there were significantly reduced freezing levels in PTZ + Veh animals when compared with Sham + Veh controls (n = 11–15/group). The PTZ + Wort group exhibited significantly reduced freezing levels as compared with the Sham + Veh group (n = 11–15/group). However, significantly elevated freezing levels were present in PTZ + Wort animals relative to PTZ + Veh group (n = 11–15/group). Wort treatment did not affect the freezing levels in Sham animals. Representative Western blottings (left panels) and quantifications (right panels) depicting P-S6 (F) and P-Akt (G) immunoreactivity from whole hippocampal homogenates of animals from all treatment groups 1 h following induction are shown. F, There were significantly elevated P-S6 levels in the PTZ + Veh animals relative to Sham + Veh controls (n = 3–6/group). The PTZ + Wort group displayed significantly reduced P-S6 levels as compared with PTZ + Veh animals (n = 3–6/group). P-S6 levels were not significantly changed in Sham + Wort compared with Sham + Veh animals (n = 3–6/group). G, Similarly, in PTZ + Veh animals, P-Akt levels were significantly elevated relative to Sham + Veh animals (n = 3–6/group). The P-Akt levels were reduced in PTZ + Wort animals the relative to PTZ + Veh group (n = 3–6/group). There was no significant difference in P-Akt levels between Sham + Wort and Sham + Veh animals (n = 3–6/group). All data are represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).
- Figure 4.
A single PTZ-induced seizure leads to dendritic spine alterations in hippocampal dendrites with partial rescue using Wort. Brains were collected 3 h following a single generalized seizure and images of hippocampal area CA1 secondary and tertiary dendrites were obtained. A, High-magnification representative photomicrographs of dendrites in Sham and PTZ animals treated either with Veh or Wort. Mature spines are observed in Sham + Veh and Sham + Wort animals (red arrowheads), while dendritic spines from animals in the PTZ + Veh and PTZ + Wort conditions display immature shaped spines (red arrows). Wort treatment reduced the number of visible spines in both Sham and PTZ animals. Less immature spines are observed in PTZ + Wort animals. B, Analyses revealed that a single generalized seizure resulted in a significant reduction of the number of spine protrusions per micrometer relative to Sham + Veh animals (n = 6 animals/group, n = 4–6 neurons/animal, n = 94–113 dendrites/group). In the PTZ + Wort condition, the number of protrusions per micrometer was significantly reduced relative to Sham + Veh controls and further reduced relative to PTZ + Veh animals (n = 3–6 animals/group, n = 4–6 neurons/animal, n = 113–135 dendrites/group). There was also a significant reduction in Sham + Wort relative to Sham + Veh (n = 3 animals/group, n = 4–6 neurons/animal, n = 94 dendrites/group). C, In PTZ + Veh animals, there was a significant increase in the LWR relative to Sham + Veh controls of those spines that remained (n = 6 animals/group, n = 4–6 neurons/animal, n = 94–113 dendrites/group). The LWR was significantly reduced in PTZ + Wort animals as compared with the PTZ + Veh condition (n = 3–6 animals/group, n = 4–6 neurons/animal, n = 113–135 dendrites/group). No significant differences in LWR were found between Sham + Wort and Sham + Veh animals (n = 3–6 animals/group, n = 4–6 neurons/animal, n = 113–135 dendrites/group). D, Further analyses of spine morphology revealed that in the PTZ + Veh experimental group, there was a significant increase in the number of immature spines per micrometer relative to Sham + Veh animals (n = 6 animals/group, n = 4–6 neurons/animal). Wort treatment blocked the seizure-induced increase in immature spines per micrometer in PTZ + Wort when compared with PTZ + Veh condition (n = 3–6 animals/group, n = 4–6 neurons/animal, n = 113–135 dendrites/group). In Sham + Wort animals, there was no significant difference in the number of immature spines per micrometer relative to Sham + Veh animals (n = 6 animals/group, n = 4–6 neurons/animal). E, Finally, in PTZ + Veh animals, there was a significant reduction in the number of mature spines per micrometer when compared with Sham + Veh controls (n = 6 animals/group, n = 4–6 neurons/animal, n = 94–113 dendrites/group). There was no significant difference in the number of mature spines per micrometer in PTZ + Wort as compared with PTZ + Veh animals indicating Wort did not block the seizure-induced decrease in mature spines (n = 3–6 animals/group, n = 4–6 neurons/animal, n = 94–135 dendrites/group). There was no effect of Wort on the number of mature spines per micrometer in Sham + Wort compared with Sham + Veh animals (n = 3–6 animals/group, n = 4–6 neurons/animal, n = 94–135 dendrites/group). Data represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).
- Figure 5.
Seizure-induced long-term contextual and cued memory deficits and PI3K/Akt/mTOR hyperactivation are transient. A, Timeline of seizure induction and FC protocol is shown. A third group of animals (cohort 3) was induced as before but trained 16 h after seizure in FC. Twenty-four hours after the training (40 h after seizure), the animals were tested for long-term contextual and cued memory. B, Contextual long-term memory (left panel). When tested at 40 h after seizure, both Sham and PTZ animals displayed elevated freezing levels with no significant difference between groups (n = 11–12/group). Cued long-term memory (right panel). There was no significant difference between the Sham and PTZ animals in the Pre CS condition (n = 11–12/group). During the CS presentation, both Sham and PTZ animals displayed significantly elevated freezing levels relative to the Pre CS condition, and there was no significant difference between the two treatment groups (n = 11–12/group). C, Representative Western blotting from whole hippocampi and quantifications of P-S6 and P-Akt (left and right blot panels, respectively) at 16 h following a single seizure are shown. The bar graphs from the analysis reveal that at 16 h following a single generalized seizure there was no significant difference in the P-S6 and P-Akt levels in PTZ animals relative to Sham controls (n = 5/group). Data represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).
Tables
Figure/panel Assay Graphed residuals (Y/N) Normality analysis Probability of the goodness of fit (%) Data distribution 2A Training for short-term memory No (graphed and analyzed individual scatterplot) Nonlinear regression with Akaike's Information criterion 98.75 Parametric 2B Training for long-term memory No (graphed and analyzed individual scatterplot) Nonlinear regression with Akaike's information criterion 99.94 Parametric 3B Contextual short-term memory Yes Nonlinear regression with Akaike's information criterion 99.96 Parametric 3C, left Cued short-term memory pre CS Yes Nonlinear regression with Akaike's information criterion 99.98 Parametric 3C, right Cued short-term memory CS Yes Nonlinear regression with Akaike's information criterion 99.97 Parametric 3D Contextual long-term memory Yes Nonlinear regression with Akaike's information criterion 99.91 Parametric 3E, left Cued long-term memory pre CS Yes Nonlinear regression with Akaike's information criterion 99.86 Parametric 3E, right Cued long-term memory CS Yes Nonlinear regression with Akaike's information criterion 99.96 Parametric 3F Western blotting of P-S6 Yes Nonlinear regression with Akaike's information criterion 99.97 Parametric 3G Western blotting of P-Akt Yes Nonlinear regression with Akaike's information criterion 99.98 Parametric 4B Protrusions per micrometer Yes Nonlinear regression with Akaike's information criterion 98.58 Parametric 4C LWR Yes Nonlinear regression with Akaike's information criterion 99.85 Parametric 4D Immature spines per micrometer Yes Nonlinear regression with Akaike's information criterion 98.91 Parametric 4E Mature spines per micrometer Yes Nonlinear regression with Akaike's information criterion 86.15* Parametric 5B, left Contextual long-term memory 16-h training Yes Nonlinear regression with Akaike's information criterion 96.03 Parametric 5B, right Cued long-term memory No (graphed and analyzed individual scatterplot) Nonlinear regression with Akaike's information criterion 99.94 Parametric 5C, right Western blotting of P-S6 and P-Akt Yes Nonlinear regression with Akaike's information criterion (for S6 only), 95.53 Parametric Yes Nonlinear regression with Akaike's information criterion (for AKT only) 98.17 *Because the probability was lower than 90%, an extra sum-of-squares test was subsequently run, which reported that it is not valid to reject the null hypothesis (i.e., the data fit the model).
Figure/panel Assay Statistical test F(df, error value), p value Post hoc analysis/correction 2A Training for short-term memory Two-way repeated measures ANOVA Interaction: F(1,12) = 3.446, p = 0.0881Time: F(1,12) = 42.49, p < 0.0001Experimental group: F(1,12) = 1.686, p = 0.2185 Holm-Sidak's multiple comparisons test 2B Training for long-term memory Two-way repeated measures ANOVA Interaction: F(1,16) = 1.986, p = 0.1779Time: F(1,16) = 88.27, p < 0.0001Experimental group: F(1,16) = 0.4603, p = 0.5072 Holm-Sidak's multiple comparisons test 3C, left Cued short-term memory pre CS Ordinary one-way ANOVA F(3,46) = 16.69, p = 0.0100 Holm-Sidak's multiple comparisons test 3C, right Cued short-term memory CS Ordinary one-way ANOVA F(3,43) = 16.69, p < 0.0001 Holm-Sidak's multiple comparisons test 3D Contextual long-term memory Ordinary one-way ANOVA F(3,46) = 16.72, p < 0.0001 Holm-Sidak's multiple comparisons test 3E, left Cued long-term memory pre CS Ordinary one-way ANOVA F(3,46) = 16.69, p = 0.0100 Holm-Sidak's multiple comparisons test 3E, right Cued long-term memory CS Ordinary one-way ANOVA F(3,45) = 20.39, p < 0.0001 Holm-Sidak's multiple comparisons test 3F Western blotting of P-S6 Ordinary one-way ANOVA F(3,13) = 9.391, p = 0.0014 Holm-Sidak's multiple comparisons test 3G Western blotting of P-Akt Ordinary one-way ANOVA F(3,11) = 5.058, p = 0.0192 Holm-Sidak's multiple comparisons test 4B Protrusions per micrometer Ordinary one-way ANOVA F(3,613) = 13.55, p < 0.0001 Holm-Sidak's multiple comparisons test 4C LWR Ordinary one-way ANOVA F(3,645) = 14.45, p = 0.0009 Holm-Sidak's multiple comparisons test 4D Immature spines per micrometer Ordinary one-way ANOVA F(3,609) = 4.505, p = 0.0039 Holm-Sidak's multiple comparisons test 4E Mature spines per micrometer Ordinary one-way ANOVA F(3,613) = 4.845, p = 0.0023 Holm-Sidak's multiple comparisons test 5B, left Contextual long-term memory 16-h training Unpaired Student's t test t(18.31) = 0.3066, p = 0.7626 Welch's correction 5B, right Cued long-term memory Two-way repeated measures ANOVA Interaction: F(1,21) = 0.5235, p = 0.4773Time: F(1,21) = 133.2, p < 0.0001Experimental group: F(1,21) = 0.3028, p = 0.5879 Holm-Sidak's multiple comparisons test 5C, right Western blotting of P-S6 and P-Akt Unpaired Student's t test (for S6 only) t(6.257) = 0.5123, p = 0.6260 Welch's correction Unpaired Student's t test (for AKT only) t(7.997) = 1.713, p = 0.1250 Welch's correction
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