Article Figures & Data
Figures
- Figure 1.
Spontaneous seizures occur within the first month after SE. A, Description of SE protocol. Expansion below to the left illustrates the only difference between the treatment groups was an injection of kainic acid (post-SE) or vehicle (control). All rats, including controls, received an injection of pentobarbital. After status, post-SE rats underwent a variable length latent period, and then chronic spontaneous seizures begin. Rats were euthanized one to two months after the day of SE induction and used for physiology or immunohistochemistry experiments. B, Example EEG from video-EEG monitoring of post-SE rat implanted with depth electrodes targeting the dorsal, intermediate, and ventral hippocampus. The first seizures were subclinical and occurred on day 8. The first electrographic seizures that also had a behavioral correlate, which were confirmed by video monitoring, occurred on day 12. The electrographic signature of these seizures is shown at the top. Below, the seizure frequency is shown through day 45 post-SE. C, Representative EEG from an animal implanted with subdural electrodes positioned above the left and right parietal association cortex (L PA and R PA, respectively) and right frontal cortex (R FC). Video-EEG monitoring showed that before kainic acid injections, seizures were never observed. Pre-SE traces were taken from a period of quiet wakefulness, and traces to the right (post-SE) show the first observed convulsive seizure 13 d post-SE. Four rats were equipped with subdural electrodes and monitored 3 d/week for 8 h/d for at least the first two months post-SE. One animal was monitored continuously (from B, filled diamond). Seizures were observed in the first month post-SE in all rats. The day of the first observed convulsive seizure for animals is plotted. D, Days after injection (post-SE or control) is plotted for animals used in these studies.
- Figure 2.
Slices were collected from identifiable and distinct regions of the hippocampus. A, Preparation of all slices used in the following experiments targeted either the dorsal or the ventral hippocampus. To the left, a schematic of the blocking cuts used to collect slices from the dorsal (top) or ventral (bottom) hippocampus. To the right, representative sections show differences in hippocampal microarchitecture at either end of the dorsoventral axis. Note the distinctive difference in the shape of the dentate granule cell layer. B, Dorsoventral location of slices were mapped on to the longitudinal axis of the hippocampus post hoc. Left, Representative images from above are overlaid with solid and dotted lines. These lines reflect measurements made from transverse hippocampal sections. A ratio of the length of the lines (solid/dotted) in hippocampal subregions CA1 (green), CA3 (orange), and DG (yellow) were put into the statistical model described in the text to estimate dorsoventral location. Right, Summary of predicted slice location along the dorsoventral axis. Symbols reflect location (e.g., circles are dorsal, and triangles are ventral hippocampus). Data from control rats are presented in black, and data from post-SE rats are presented in red. This color scheme is consistent throughout the manuscript.
- Figure 3.
Dorsoventral difference in firing output is absent post-SE. A, To the left the schematic shows the recordings location. To the right representative action potential trains evoked from the natural resting membrane potential with an 800-ms-long 250-pA current injection. B, The firing intensity, which is the number of action potentials generated as a function of the amplitude of injected current, is plotted for current steps between 0 and 500 pA. The firing intensity obtained from whole-cell recordings of dorsal (circles) and ventral (triangles) CA1 pyramidal neurons from control animals are plotted. When we compared these group in post hoc comparisons we found that they were different for all current steps above 150 pA. C, To compare the effect of epilepsy within regions, we plotted the firing intensity of dorsal CA1 neurons from control (black) and post-SE (red) rats. D, Firing intensity of ventral CA1 neurons from control and post-SE rats were not statistically different at any current injection. E, Firing intensity of dorsal and ventral CA1 neurons from post-SE rats. Data are expressed as the mean ± SEM. Statistical significance (*) is defined as p < 0.05.
- Figure 4.
Reduced ISI contributes to increased firing in dorsal CA1 neurons post-SE. A, Representative trains of 8–11 action potentials from dorsal CA1 neurons from control and post-SE groups. Trains are evoked from the resting membrane potential with a variable amplitude current injection. The traces are overlaid at the bottom, which allows for direct comparison of trace features. B, Threshold, plotted as a function of spike number in the train, progressively increases, but was not different between the two groups. C, Maximum rate of rise was also not different between the two groups. D, Action potential amplitude was also not different between the two groups. E, The amplitude of the fAHP immediately following repolarization was also measured. The amplitude was not different between the control and post-SE dorsal neurons. F, ISI was measured between each action potential. Control dorsal neurons had a characteristic delay in the middle of the train, which was absent post-SE. *p < 0.05.
- Figure 5.
Input resistance is increased in dorsal CA1 neurons post-SE. A, Dorsal and ventral cells had different resting membrane potentials in recordings from control rats. B, Resting membrane potentials of dorsal and ventral CA1 neurons from post-SE rats are plotted. C, Current injections delivered to neurons were 800-ms-long steps ranging from –150 to 50 pA. Representative voltage traces from dorsal CA1 neurons from control (black) and post-SE (red) groups are shown. Neurons were held at –65 mV. D, Representative voltage traces recorded from ventral CA1 neurons in control and post-SE groups are shown. E, Dorsal-ventral comparisons of input resistance from control rats are plotted. F, The input resistance of dorsal CA1 neurons from control and post-SE groups are plotted. G, Within ventral CA1, measurements of input resistance from control and post-SE neurons were plotted. H, Dorsal-ventral comparisons of input resistance from post-SE rats are plotted. *p < 0.05.
- Figure 6.
Dendritic input resistance is increased in dorsal CA1 neurons post-SE. A, B, Representative voltage traces from 800-ms-long current steps from –150 to 50 pA in dorsal (A) and ventral (B) CA1 neurons from control (black) and post-SE (red) groups. Dendrites were held at –65 mV. C, Input resistance was calculated from families of traces collected at –75, –70, –65, and –60 mV for dorsal and ventral neurons from control animals. Inset shows recording location. D, The input resistance of dorsal dendrites from control and epileptic animals were significantly different from each other. E, Epilepsy as a factor did not have a significant effect in describing the variance of the input resistances of ventral dendrites. F, The input resistance of dorsal and ventral neurons from post-SE animals are plotted. *p < 0.05.
- Figure 7.
Cellular morphology unchanged in dorsal and ventral CA1 neurons post-SE. A, Representative morphologic reconstructions of filled dorsal CA1 neurons from control and post-SE groups. B, Branching pattern of dorsal neurons quantified with Sholl analysis where each concentric circle increased by 20.3 µm. C, Total dendritic length in dorsal neurons from both control and post-SE groups are plotted. D, Representative neuron tracings of ventral CA1 neurons from control and post-SE groups. E, Sholl analysis quantified branching of the dendritic arbor in ventral CA1 neurons. F, The dendritic length in ventral CA1 neurons in control and post-SE were not statically different.
- Figure 8.
Reduced ISI in dorsal CA1 neurons post-SE cannot be explained by a reduction in M channel expression. A–D, Action potential trains containing 8–11 spikes were analyzed as in Figure 4. Example spike trains evoked from –60 mV are shown before and after (gray) 10 µM XE991. Summary graph shows ISI duration versus position in spike train before (solid line/closed circles) and after XE991 (dashed line/open circles). A, In dorsal CA1 neurons from control and post-SE rats, the duration of the ISI increases throughout the train. B, The SFA, calculated as a ratio of the first to the sixth spike, was not significantly different between control and post-SE dorsal neurons; however, there was a significant effect of XE. C, In ventral CA1 neurons from control and post-SE rats, the duration of the ISI remains at ∼100 ms throughout the train. There are no differences between control and post-SE conditions. D, The SFA was close to 1, and XE991 had a minimal effect on these neurons. There was no difference between control and post-SE ventral neurons. E, Representative traces to the left are, from top to bottom, of injected current (–50 pA, 500-ms step and a ±50 pA, 1- to 15-Hz chirp stimulus), and representative voltage responses for dorsal CA1 neurons from control and post-SE groups at –35 mV. Arrow shows peak resonance frequency. To the right, an overlay of the impedance amplitude profiles from the representative traces with dotted lines representing the peak resonance frequency. F, Peak resonance frequency at depolarized membrane potentials is graphed. Control and post-SE dorsal neurons were not different.
- Figure 9.
Kv7.2 immunoreactivity does not change post-SE. A, B, D, E, All representative images follow the same format. Upper left, A transverse hippocampal section with a nuclear stain provides local histologic landmarks. Upper right, Kv7.2 staining in the same slice. The blue box shows the region of CA1 expanded below. The yellow box represents the quantification area. Bottom, Zoomed in view of CA1 with subunit stain and overlay of quantification area. Scale bars = 500 µm. A, Representative section from the dorsal hippocampus with Kv7.2 staining from a control rat. B, Representative section from the dorsal hippocampus with Kv7.2 staining from a post-SE rat. C, Group data of the mean gray value of region in stratum oriens of dorsal hippocampal slices from control and post-SE groups. These data are not statistically different. D, Kv7.2 staining in the ventral hippocampus of a control rat. E, Kv7.2 staining in the ventral hippocampus of a post-SE rat. F, Summary data showing the average gray value in control and post-SE groups. These data are not statistically different from one another.
- Figure 10.
The functional expression of GIRK channels is unaltered in dorsal and ventral neurons post-SE. A, With the bath application of 50 µM barium, the membrane potential increased in both control and post-SE groups. The barium induced depolarization was 7.6 ± 1.2 mV in controls and 7.0 ± 1.2 mV in the post-SE group. This difference was not statistically significant (unpaired t test, p = 0.69). B, Representative voltage traces held at –65 mV from which the input resistance was calculated in ACSF (baseline) and barium conditions in cells from control and post-SE groups. C, Somatic input resistance at –65 mV before and after application of barium in control and post-SE. Relative to baseline, the input resistance in controls increased by 33.0 ± 9.2%, and post-SE, the increase was 40.3 ± 4.4%. The change in input resistance was not statically different between the two groups. D, Bath application of 50 µM barium caused the membrane potential of ventral CA1 neurons from both groups to depolarize. Summary graphs show the change in the control and post-SE groups. The change in membrane potential was not different between the two groups. E, Representative voltage traces held at –65 mV from which the input resistance was calculated in ACSF (baseline) and barium conditions in control (black) and post-SE (red). F, Barium caused the steady state input resistance to increase in ventral CA1 neurons from both control and post-SE groups. The percentage change relative to baseline was 18.1 ± 3.5% for controls and 33.7 ± 10.8% post-SE. This difference was not statistically significant.
- Figure 11.
Expression of GIRK2 subunit is unchanged post-SE. A, B, D, E, All representative images follow the same format. Upper left, Transverse slice from dorsal hippocampus with the nuclear stain, Hoechst 33342, from control group. Upper right, Representative hippocampal staining of GIRK2. The blue box shows the portion of CA1 expanded below. The yellow shaded region shows the region selected for quantification from the alveus to the fissure in both channels. Bottom, GIRK2 staining in CA1, where the lighter shade of gray reflects more immunoreactivity for GIRK2 protein. Staining is evident in the somatic layer (S.P.) and dendritic layers. Scale bars = 500 µm. A, Representative section from the dorsal hippocampus with GIRK2 staining from a control rat. B, Representative section from the dorsal hippocampus with GIRK2 staining from a post-SE rat. C, Quantification of average grayscale pixel intensity along the length of the somatodendritc axis on dorsal CA1. Since the radial length can differ between sections, the lengths were normalized and binned into 20 segments. Dotted lines reflect transitions between layers abbreviated S.O. (stratum oriens), S.P. (stratum pyramidale), S.R. (stratum radiatum), and S.L.M. (stratum lacunosum moleculare). Comparisons between equivalent radial locations were tested between control and post-SE group data. D, GIRK2 staining in the ventral hippocampus of control rat. E, GIRK2 staining in the ventral hippocampus of a post-SE rat. F, Quantification along the normalized length of the somatodendritic/radial axis in ventral CA1. Equivalent radial locations were compared between control and post-SE group data.
- Figure 12.
Resonance frequency is reduced in dorsal dendrites post-SE. A, C, E, G, left, Representative voltage responses to a 15-Hz chirp stimulus obtained from whole-cell recordings from control (upper, black) and post-SE (lower, red) cells. Right, Plot of impedance amplitude as a function of frequency for voltage traces. Dotted line shows the peak frequency. A, Representative recordings from the soma of dorsal CA1 neurons. B, Summary graph showing the peak resonance frequency values obtained from dorsal somatic recordings from control and post-SE groups. These groups were not statically different from one another. C, Representative recordings from the apical dendrite of dorsal CA1 neurons. D, Summary of data collected from the dendrite of dorsal CA1 neurons showing the peak resonance in control and post-SE groups. These groups were statistically different. E, Representative responses at the soma of ventral CA1 neurons in both groups. F, Summary graphs showing the group data for control and post-SE conditions. G, Representative recordings at the dendrite of ventral CA1 neurons. H, Summary graphs comparing the group data for control and post-SE conditions. *p < 0.05.
- Figure 13.
Reduced sensitivity to the HCN channel blocker, ZD7288, in dorsal dendrites post-SE. A, Voltage responses at the dendrite of dorsal CA1 neurons were measured from –70 mV under baseline conditions, ACSF, and after bath application of 10 µM ZD7288. B, The steady state input resistance increased after ZD was introduced in both control and post-SE groups. C, The increase in input resistance was much larger in controls. D, The amplitude of the rebound depolarization was plotted as a function of the membrane potential at the end of the current step. The slope of this relationship was plotted under baseline, ACSF, conditions and then after ZD7288 for both treatment groups. In dorsal CA1 neurons post-SE, the rebound slope was significantly reduced. E, Representative traces from dendrites of ventral CA1 neurons under baseline conditions, ACSF, or after bath application of 10 µM ZD7288. F, The dendritic input resistance of ventral CA1 neurons increased after ZD7288. G, The increase in input resistance relative to baseline was not different between the groups. H, The rebound slope was reduced with application of ZD, but there was no difference between the control and post-SE groups. *p < 0.05.
- Figure 14.
Reduced expression of HCN1 subunit in distal dendritic layer post-SE. A, B, D, E, Upper left, Representative hippocampal section with the nuclear stain. Upper right, HCN1 staining. The blue box shows the portion of CA1 expanded below. The yellow box shows the region selected for quantification from the alveus to the fissure. Bottom, HNC1 staining in CA1, where the lighter shade of gray reflects more immunoreactivity for HCN1 protein. The most prominent staining is in the distal dendrites of S.L.M. Scale bars = 500 µm. B, Representative section shown of dorsal hippocampal section from post-SE rat. C, Quantification along the length of the somatodendritc axis on dorsal CA1. Dotted lines reflect transitions between layers. Comparisons between equivalent radial locations were tested between control and post-SE group data. D, HCN1 staining in the ventral hippocampus of a control rat. E, HCN1 staining in the ventral hippocampus of a post-SE rat. F, Quantification along the normalized length of the somatodendritic axis in ventral CA1. Equivalent radial locations were compared between control and post-SE group data. *p < 0.05.
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