Article Figures & Data
Figures
- Figure 1.
Kcc2/Crh KO male mice exhibit an exaggerated, seizure-induced activation of the HPA axis. A, Circulating corticosterone concentration collected 2 h after intraperitoneal injections of either saline or KA in WT and Kcc2/Crh KO male mice. B, Circulating corticosterone levels were quantified from serum collected 24 h following pilocarpine-induced SE in WT and Kcc2/Crh KO male mice. n = 18 (WT saline); 5 (Kcc2/Crh KO saline); 17 (WT KA); 8 (Kcc2/Crh KO KA); 12 (WT pilocarpine); 9 (Kcc2/Crh KO pilocarpine). Error bars represent ±SEM.
- Figure 2.
Chronically epileptic Kcc2/Crh KO male mice exhibit increased vulnerability to negative affective states compared with WT control mice. Top, Experimental paradigm illustrating the timeline of intrahippocampal injections and behavioral testing. Tests for avoidance (A–D), learned helplessness (E, F), anhedonia (G), and goal-directed (H) behaviors were tested in control and chronically epileptic male WT and Kcc2/Crh KO mice. A, B, The average total time spent in the center (A) and total distance traveled in the center of the OF test (B). C, D, The average total time spent (C) and total distance traveled (D) in the light arena of the LD setup. E, F, The histograms depict the average total time spent immobile (E) and the latency to the first bout of immobility (F) in the FST. G, The average percent sucrose preference measured over the course of a 7 d SPT paradigm. H, The average percentage of unshredded nestlet material was weighed daily for 1 week. N = 11–20 per group. Error bars represent SEM. OF, open field; LD, light/dark; FST, forced swim test; SPT, sucrose preference test; NST, nestlet-shredding test. The proportion of chronically epileptic Kcc2/Crh KO mice that exhibit increased vulnerability to negative affective states compared with chronically epileptic (vIHKA) WT mice is provided in Extended Data Figure 2-1.
- Figure 3.
Chronically epileptic females with HPA axis dysfunction exhibit increased incidence of negative affective states. Top, Experimental paradigm illustrating the timeline of intrahippocampal injections and behavioral testing. Tests for avoidance (A–D), learned helplessness (E, F), anhedonia (G), and goal-directed (H) behaviors were tested in control and chronically epileptic female WT and Kcc2/Crh KO mice. A, B, The total amount of the time spent in the center of the OF (A) and the total distance traveled in the OF (B) were measured in the control and chronically epileptic female WT and Kcc2/Crh KO mice. C, D, The total time spent (C) and the total distance traveled (D) in the light arena of the LD box was measured and quantified. E, F, The total time spent immobile (E) and the latency to immobility (F) in a 6 min FST paradigm were measured and quantified. G, The average sucrose water consumed over a 7 d period was measured for the SPT paradigm. H, Chronically epileptic and control WT and Kcc2/Crh KO mice were singly housed and given a piece of unshredded nestlet. The percentage of nestlet that remained unshredded was weighed daily over the course of 7 d. N = 10–14 mice per group. Error bars represent SEM. OF, open field; LD, light/dark; FST, forced swim test; SPT, sucrose preference test; NST, nestlet-shredding test.
- Figure 4.
HPA axis dysfunction does not worsen spontaneous seizure activity in chronic epilepsy in males. Top, Experimental paradigm illustrating the timeline of intrahippocampal injections with treatment and EEG/LFP recordings. The seizure detection pipeline is described in Extended Data Figure 4-1. A, Representative recorded seizure from hippocampal local field potential. Orange squares indicate time magnified 5 s traces. B, Example detected hippocampal seizures which were concatenated from a WT and a Kcc2/Crh KO (KO) mouse. C–E, Mean (±SEM) number of daily seizure occurrences (C), seizure duration (D), and seizure burden (E) for chronically epileptic WT, Kcc2/Crh KO, and Kcc2/Crh KO mice treated with RU486, a glucocorticoid receptor antagonist. F–H, Average number of daily seizures (F), seizure durations (G), and seizure burden (H) for chronically epileptic Kcc2/Crh KO mice bilaterally injected with hM4D(Gi)-DREADDs in the PVN. Graphs F–H depict mean seizure activity collected via EEG recordings that were made prior to CNO administration (baseline), during CNO administration (+CNO), and during a week free of CNO administration (−CNO). Seizure burden was calculated by multiplying the total number of seizures a mouse exhibited by their average seizure duration and dividing this value by the total EEG recording hours. Error bars represent SEM. The impact of HPA axis dysfunction on neuropathological features of epilepsy in males is provided in Extended Data Figure 4-2.
- Figure 5.
HPA dysfunction does not alter seizure severity in chronically epileptic female Kcc2/Crh KO mice. Top, Experimental paradigm illustrating the timeline of intrahippocampal injections with treatment and EEG/LFP recordings. A, A representative EEG trace showing concatenated seizure events detected from WT and Kcc2/Crh KO female mice recorded from hippocampal local field potential. B–D, The seizure frequency (B), average seizure duration (C), and overall seizure burden (D) quantified for chronically epileptic female WT, Kcc2/Crh KO, and Kcc2/Crh KO mice treated with the glucocorticoid receptor antagonist, RU486. F–H, The daily seizure frequency (E), mean seizure duration (F), and overall seizure burden (G) quantified from chemogenetically manipulated chronically epileptic female Kcc2/Crh KO mice. Each period (baseline, with CNO, and without CNO treatment) was measured over a 7 d period. Seizure burden was calculated by multiplying the total number of seizures a mouse exhibited by their average seizure duration and dividing this value by the total EEG recording hours. Error bars represent SEM. The impact of HPA axis dysfunction on neuropathological features of epilepsy in females is provided in Extended Data Figure 5-1.
- Figure 6.
HPA axis dysfunction in chronically epileptic Kcc2/Crh KO mice increases SUDEP incidence in males. Top, Experimental paradigm illustrating the timeline of intrahippocampal injections with treatment and EEG/LFP recordings for SUDEP monitoring. A–C, Percent survival following vIHKA-induced SE in chronically epileptic WT and Kcc2/Crh KO male mice (A), in chronically epileptic Kcc2/Crh KO mice treated with the glucocorticoid receptor antagonist, RU486 (B), and in chronically epileptic Kcc2/Crh KO mice expressing Gi DREADDs and given CNO (C). SUDEP, sudden unexpected death in epilepsy; WT, wild-type; SE, status epilepticus. D–F, Percent survival after kainic acid-induced SE in chronically epileptic female WT and Kcc2/Crh KO female mice (D), in chronically epileptic female Kcc2/Crh KO mice treated with and without the glucocorticoid receptor antagonist, RU486 (E), and in chronically epileptic Kcc2/Crh KO mice treated with Gi DREADDs and given CNO via drinking water to reduce HPA axis activity (F). Upward ticks on survival plots indicate the time that mice were killed. For visual comparisons, the Kcc2/Crh survival plots have been replotted in each subpanel (same dataset) across male (A–C) and across female (D–F) mice.
- Figure 7.
Chronic HPA axis dysfunction may increase SUDEP risk in PWE. A, Experimental schematic. B, Postmortem analysis of circulating CORT, (C) CRH, (D) epinephrine, (E) norepinephrine (NE), and (F) E/NE ratio in control subjects, PWE, and subjects who died of SUDEP. Error bars represent SEM. Data were analyzed using a one-way ANOVA. PWE, person(s) with epilepsy; SUDEP, sudden unexpected death in epilepsy.
Extended Data
Extended Data
Download Extended Data, DOCX file.
Figure 2-1
A greater proportion of the chronically epileptic Kcc2/Crh KO mouse population exhibit increased vulnerability to negative affective states compared to chronically epileptic (vIHKA) WT mice. Smoothed population distributions (top) along with raw histogram distribution (bottom) of performance in the Open Field (A-B), Light Dark box (C-D), and Forced Swim Test (E-F) between chronically epileptic WT (left) and Kcc2/Crh KO (right) mice. In each smoothed plot, the lighter color represents the underperforming, more vulnerable population while the black distribution plots represent the resilient groups. # denotes instances where only one peak was detected, so population distributions were delineated by the mean of the data. Download Figure 2-1, TIF file.
Figure 4-1
Seizure detection pipeline. (A) Left - Datasets used to train and test the seizure detection algorithm, Upper Right – Example traces for no-seizure and seizure (5 second periods), Bottom Right – Minimum number of seizure segments for an event to qualify as a seizure. (B) Histograms with KDE plots showing the Top – number of false detected seizures per hour and Bottom – percentage of detected seizures; Pink line denotes performance of chosen method. (C) Scatterplot of percent detected seizures vs false positive rate. Pink dot indicates performance of chosen method. Detailed pipelines can be found at https://github.com/neurosimata/seizy. Download Figure 4-1, TIF file.
Figure 4-2
HPA axis dysfunction worsens MFS in male chronically epileptic mice. (A) Representative coronal sections of the hippocampus collected from control and chronically epileptic adult, male WT and Kcc2/Crh KO mice and stained with ZnT3 to quantify MFS. White arrow indicates mossy fiber sprouting. In some slices, we observed that dentate completely lost structural integrity as assessed by ZnT3 staining; example indicated by a red outline (B) The mean (±SEM) percent change in MFS was quantified in the ipsilateral hemisphere and normalized to the mean percent change in MFS of the contralateral hemisphere of chronically epileptic WT and Kcc2/Crh KO mice. Dotted black line indicates no MFS in animals that received saline injection. In slices with complete loss of structural integrity, MFS was quantified as the full dentate length. Those slices are indicated by colored filled dots on the graph. (C-D) Representative sections stained with NeuN to visualize DGCD in WT (C) and Kcc2/Crh mice (D). Pink outlines were automatically generated through Cell Profiler and indicate cells where NeuN and DAPI colocalize. (E) The mean (±SEM) number of adjoining neighboring neuronal cells was quantified for the ipsilateral hemisphere and normalized to the mean number of immediate neighboring neuronal cells on the contralateral hemisphere for both control and chronically epileptic WT and Kcc2/Crh KO mice. (F) The total number of cells within the manually defined dentate gyrus area was quantified on the ipsilateral hippocampal hemisphere and normalized to the cell density of the non-injected, contralateral hippocampal hemisphere. n = brain slice sections. Error bars represent ± SEM. WT, wild type; Sal, saline; KA, kainic acid; DGCD, dentate granule cell dispersion; Norm, normalized. Download Figure 4-2, TIF file.
Figure 5-1
HPA axis dysfunction worsens MFS in female chronically epileptic mice. (A) Representative coronal sections of the hippocampus collected from control and chronically epileptic adult, female WT and Kcc2/Crh KO mice and stained with ZnT3 to quantify MFS. White arrows indicate MFS. (B) The mean (±SEM) percent change in MFS was quantified in the ipsilateral hemisphere and normalized to the mean percent change in MFS of the contralateral hemisphere of chronically epileptic WT and Kcc2/Crh KO mice. Dotted black line indicates no MFS in animals that received saline injection. Representative sections stained with NeuN to visualize DGCD in WT (C) and Kcc2/Crh KO mice (D). Pink outlines were automatically generated through Cell Profiler and indicate cells where NeuN and DAPI colocalize. (E) The mean (±SEM) number of adjoining neighboring neuronal cells was quantified for the ipsilateral hemisphere and normalized to the mean number of immediate neighboring neuronal cells on the contralateral hemisphere for both control and chronically epileptic WT and Kcc2/Crh KO female mice. (F) The total number of cells within the manually defined dentate gyrus area was quantified on the ipsilateral hippocampal hemisphere and normalized to the cell density of the non-injected, contralateral hippocampal hemisphere. n = brain slice sections. Error bars represent ± SEM. WT, wild type; Sal, saline; KA, kainic acid; DGCD, dentate granule cell dispersion; Norm, normalized. Download Figure 5-1, TIF file.
In this issue
