Figures and data
Mice have resting tachycardia shortly after vIHKA injection.
A) Resting HR is no different between WT (gray, Kcc2f/f) and mice with hyperactive stress circuits (blue, Kcc2/Crh) prior to hippocampal injection. B) Example HR traces from mice with and without hyperactive stress circuits with telemeters that were monitored weekly following vIHKA injection. C) Resting HR was significantly elevated 7 days after vIHKA injection in both mice with (*p=0.0190, Mixed Effects Analysis and Tukey’s Post Hoc) and without hyperactive stress circuits (*p=0.0169, Mixed Effects Analysis and Tukey’s Post Hoc). HR remained elevated in mice with hyperactive stress circuits (**p=0.0017, Mixed Effects Analysis and Tukey’s Post Hoc) but not WT, 14 days after vIHKA injection. D) Mice with hyperactive stress circuits that died following vIHKA injection are plotted individually (dashed lines). The average HR over time of mice with hyperactive stress circuits that survive to endpoint are plotted together (solid line). There appears to be no relationship between magnitude of resting tachycardia and SUDEP susceptibility. Symbols are group means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 compared to day 0 by mixed effects model and Tukey’s post-hoc test, #p < 0.05 compared to sham control by mixed effects model and Tukey’s post-hoc test.
Figure 1C- HR normalized to Day 0 (BPM)
Mice with hyperactive CRH neurons have greater ictal bradycardia than WT mice.
A) Example telemetry recordings of EEG (top) and heart rate (HR, bottom) during a spontaneous seizure in WT (left, gray) and Kcc2/Crh (right, blue) mice. Prior to seizure onset, HR began to rise (pre-ictal tachycardia, light green). During the seizure, elevated HR (ictal tachycardia, red) was interrupted by transient drops in HR (blue). Kcc2/Crh mice showed severe drops in HR during seizures. B) Qualitative representation of grouped average HR in reference to seizure termination. HR during seizures in WT (white shading) and Kcc2/Crh (Blue shading) were similar with Kcc2/Crh mice having more pronounced drops in HR right before seizure termination. C) Heatmap depicting grouped average change in HR relative to baseline in reference to seizure termination. Kcc2/Crh mice had a more pronounced drop in HR right before seizure termination. D-I, Summary data of mean ictal HR, ictal tachycardia magnitude, ictal tachycardia duration, pre-ictal tachycardia time of onset, ictal bradycardia magnitude, and ictal bradycardia duration. Kcc2/Crh mice had significantly greater magnitude (p=0.0264, unpaired t-test) and duration (p=0.0350, unpaired t-test) of ictal bradycardia compared to WT mice. Bars are group means ± SD,* p < 0.05 by unpaired t test.
Mice have blunted baroreflex sensitivity 30 days after vIHKA injection.
A) Example blood pressure (BP, top) and heart rate (HR, bottom) recordings during jugular infusion of phenylephrine (PE) in WT (Kcc2f/f) mice at 10 (middle, medium gray) and 30 (right, dark gray) days post vIHKA injection or in saline-injected sham controls (left, light gray). B) Example BP (top) and HR (bottom) recordings during jugular infusion of PE in Kcc2/Crh mice either at 10 (middle, dark blue) and 30 (right, purple) days post vIHKA injection or in saline-injected sham controls (left, light blue). In all experiments, infusion of PE caused an increase in BP and drop in HR. C) Summary data of PE-mediated increase in BP. D) Summary data of baroreflex set-point (V50) and E) Summary data of baroreflex sensitivity (slope) that was determined via the slope of resulting curve from fit with modified Boltzmann equation (colored inset). On day 30, baroreflex sensitivity (slope) was significantly blunted (*p=0.0198, Two-way ANOVA with Tukey’s Post Hoc) and V50 (*p=0.0359, Two-way ANOVA with Tukey’s Post Hoc) significantly increased compared to day 10, independent of genotype. Bars are group means ± SD, * p < 0.05 by two-way Anova and Tukey’s post-hoc test.
Figure 3C- Phenylephrine mediated increase in BP (ΔmmHg)
Figure 3E- Slope (ΔBPM/ΔmmHg)
Figure 3D- V50 (mmHg)
vIHKA injection promotes robust time-dependent changes in paradoxical bradycardia.
A) Example blood pressure (BP, top) and heart rate (HR, bottom) recordings during jugular infusion of sodium nitroprusside (SNP) in WT (Kcc2f/f) mice at 10 (middle, medium gray) and 30 (right, dark gray) days post vIHKA injection or in saline-injected sham controls (left, light gray). B) Example BP (top) and HR (bottom) recordings during jugular infusion of SNP in Kcc2/Crh mice either at 10 (middle, dark blue) and 30 (right, purple) days post vIHKA injection or in saline-injected sham controls (left, light blue). In all experiments, infusion of SNP caused a decrease in BP. Changes in HR tended to occur in a biphasic manner, where an initial (early) increase in HR was often followed by a fall in HR (late). C) Summary data showing a significant and sustained drop in BP in collapsed groups following jugular infusion of SNP. D) Summary data of SNP-mediated change in HR, with WT mice having paradoxical bradycardia (significant decreases in HR during late phase) on day 10(@p=0.0004, †p=0.0002, #p=0.0384) and Kcc2/Crh mice having paradoxical bradycardia on both day 10(@p=0.0206, #p=0.0346) and day 30(@p=0.0010, †p<0.0001, #p<0.0001). WT mice on day 30 had blunted paradoxical bradycardia relative to WT mice on day 10 ($p=0.0100) and Kcc2/Crh mice on day 30 ($p=0.0030). E) Summary data of change in HR over change in BP shows HR changes more for a given change in BP in the late phase on day 10 (@p=0.0106) in WT mice and on day 10 (@p=0.0011, #p=0.0411) and 30 (@p=0.0094, #p=0.0247) in Kcc2/Crh mice. Symbols are group means ± SEM, † p < 0.05 compared to group baseline, # p < 0.05 compared to saline-injected sham control in the same phase, @ p < 0.05 compared to “early” in group, $ p < 0.05 compared to late WT d30; Analysis by repeated measures two-way ANOVA and Tukey’s post hoc.
vIHKA injection promotes robust time-dependent increase in BJR in mice with hyperactive CRH neurons, which contributes to a subset of SUDEP mortality.
A) Example trace showing jugular infusion of phenylbiguanide (PBG) elicits the Bezold Jarisch Reflex (BJR), which is characterized by a transient decrease in heart rate (HR). The BJR is not blocked by IP administration of atenolol, but subsequent administration of scopolamine abolishes BJR. B) Summary data (bold line is average; individuals in small gray lines, n=6) showing the BJR is significantly decreased with administration of scopolamine and atenolol compared to atenolol alone (**p=0.0023, Repeated Measures One-way ANOVA with Tukey’s Post Hoc) or baseline (*p=0.0441, Repeated Measures One-way ANOVA with Tukey’s Post Hoc). C) Example ECG traces (bottom) with derived HR (top) during jugular infusion of PBG to elicit the BJR in WT (Kcc2f/f, gray) at 10 (medium gray) and 30 (dark gray) days post vIHKA injection or in saline-injected sham controls (light gray). D) Example ECG traces(bottom) with derived HR (top) during jugular infusion of PBG to elicit the BJR in mice with hyperactive CRH neurons (Kcc2/Crh) at 10 (dark blue) and 30 (purple) days post vIHKA injection or in saline-injected sham controls (light blue). E) Summary data of BJR-mediated change in HR over time. Kcc2/Crh mice have a selective exaggeration of the BJR on day 10 relative to Kcc2/Crh saline-injected sham control (***p=0.0005, Two-way ANOVA with Tukey’s Post Hoc) and WT at day 10 (***p=0.0007, Two-way ANOVA with Tukey’s Post Hoc). This exaggeration is attenuated on day 30 (**p=0.0074, Two-way ANOVA with Tukey’s Post Hoc). F) Following vIHKA injection, 9/22 mice with hyperactive stress circuits died suddenly, with a peak in mortality occurring about a week post injection. WT mice are not susceptible to SUDEP (0/12 died). Chronic treatment with methylscopolamine attenuated SUDEP mortality (3/10 died). Bars are group means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 5B- PBG mediated ΔHR (BPM)
Figure 5E- PBG mediated ΔHR over time (BPM)
vIHKA injection induces hallmark pathological features of Epilepsy in mice.
A) Representative coronal sections of the hippocampus collected from WT mice (Kcc2f/f) and mice with hyperactive stress circuits (Kcc2/Crh) at 10 and 30 days post vIHKA injection, or in sham controls that were injected with saline in the hippocampus. DAPI (blue) stained cell nuclei and was used to quantify dentate granule (DG) cell dispersion (inverse of cell density). Znt3 (green) stained zinc rich mossy fibers which, in normal conditions, project through the hilus. Znt3 was used to quantify mossy fiber sprouting (MFS), or the sprouting of mossy fibers onto other dentate granule cells in the granule cell layer (GCL) and into the molecular layer (ML). B) Summary Data showing a significant increase in DG cell dispersion on day 10 compared to saline-injected sham controls (****p<0.0001, Two-way ANOVA with Tukey’s Post Hoc), independent of genotype. On day 30, DG cell dispersion remained greater than saline-injected sham controls in collapsed groups (****p<0.0001, Two-way ANOVA with Tukey’s Post Hoc). Finally, collapsed groups had significantly increased DG cell dispersion on day 30 compared to day 10 (***p=0.0005, Two-way ANOVA with Tukey’s Post Hoc). C) Summary data showing a significant increase in MFS following vIHKA injection. MFS on day 10 (****p<0.0001, Two-way ANOVA with Tukey’s Post Hoc) and 30 (****p<0.0001, Two-way ANOVA with Tukey’s Post Hoc) was significantly increased compared to saline-injected sham controls, independent of group. Bars are group means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA with Tukey’s post hoc.
Figure 6B- Dentate Granule Cell Density (# cells/area)
