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. 2024 Mar 14;6(2):fcae052.
doi: 10.1093/braincomms/fcae052. eCollection 2024.

Peri-ictal activation of dorsomedial dorsal raphe serotonin neurons reduces mortality associated with maximal electroshock seizures

Alexandra N Petrucci  1   2   3 , Allysa R Jones  2   3 , Benjamin L Kreitlow  1   2   3 , Gordon F Buchanan  2   3
Affiliations

Peri-ictal activation of dorsomedial dorsal raphe serotonin neurons reduces mortality associated with maximal electroshock seizures

Alexandra N Petrucci et al. Brain Commun. .

Abstract

Over one-third of patients with epilepsy will develop refractory epilepsy and continue to experience seizures despite medical treatment. These patients are at the greatest risk for sudden unexpected death in epilepsy. The precise mechanisms underlying sudden unexpected death in epilepsy are unknown, but cardiorespiratory dysfunction and arousal impairment have been implicated. Substantial circumstantial evidence suggests serotonin is relevant to sudden unexpected death in epilepsy as it modulates sleep/wake regulation, breathing and arousal. The dorsal raphe nucleus is a major serotonergic center and a component of the ascending arousal system. Seizures disrupt the firing of dorsal raphe neurons, which may contribute to reduced responsiveness. However, the relevance of the dorsal raphe nucleus and its subnuclei to sudden unexpected death in epilepsy remains unclear. The dorsomedial dorsal raphe may be a salient target due to its role in stress and its connections with structures implicated in sudden unexpected death in epilepsy. We hypothesized that optogenetic activation of dorsomedial dorsal raphe serotonin neurons in TPH2-ChR2-YFP (n = 26) mice and wild-type (n = 27) littermates before induction of a maximal electroshock seizure would reduce mortality. In this study, pre-seizure activation of dorsal raphe nucleus serotonin neurons reduced mortality in TPH2-ChR2-YFP mice with implants aimed at the dorsomedial dorsal raphe. These results implicate the dorsomedial dorsal raphe in this novel circuit influencing seizure-induced mortality. It is our hope that these results and future experiments will define circuit mechanisms that could ultimately reduce sudden unexpected death in epilepsy.

Keywords: SUDEP; dorsal raphe nucleus; dorsomedial dorsal raphe nucleus; serotonin.

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Conflict of interest statement

The authors report no competing interests.

Figures

Graphical Abstract
Graphical Abstract
Figure 1
Figure 1
Terminal apnoea preceded death in TPH2-ChR2-YFP which succumbed to MES. (A) Schematic expression of the experimental timeline. (B) Immunostained coronal section, demonstrating ChR2-YFP expression, TPH2 expression and co-expression (arrows) within the DRN of a TPH2-ChR2-YFP (B) and WT (C) mouse. Box indicates location of insets (top, 5-HT cells; middle, ChR2-YFP; bottom, merge). Scale bars in B, 200 µm left, 20 µm right; Scale bars in C, 250 µm left, 50 µm right. (D) Mortality rate following optogenetic stimulation and MES in the pooled WT and TPH2-ChR2-YFP mice. TPH2-ChR2-YFP: n = 24, 10M, 14F; WT: n = 27, 12M, 15F. (E, F) Representative EEG and plethysmography traces (60 s) of TPH2-ChR2-YFP animals which either survived (E) or died (F) after optogenetic stimulation and undergoing MES. Inset in (E) demonstrates continued EKG activity during terminal apnoea in a mouse which died. Scale bars: horizontal, 5 s; vertical 1, 5, 1 μV. Error bars = SEM. (G) Coronal section demonstrating ChR2-YFP expression, TPH2 expression and c-Fos within the DRN of a TPH2-ChR2-YFP mouse following optogenetic stimulation of the DRD. Box indicates cell counting location. Scale bar = 200 µm. (H) Number of cells exhibiting TPH2 or c-Fos immunoreactivity (TPH2/c-Fos-IR) in TPH2-ChR2-YFP (n = 8) and WT mice (n = 3). P = 0.056; t = 2.201, dF = 8.776; Unpaired t-test with Welch’s correction.Dots = individual animals. Aq, aqueduct; DRD, dorsomedial dorsal raphe; DRV, dorsal raphe ventral; PLETH, plethysmography.
Figure 2
Figure 2
Seizure severity and duration were similar between pooled WT and TPH2-ChR2-YFP mice.  TPH2-ChR2-YFP: n = 24, 10M, 14F; WT: n = 25, 12M, 13F. E/F ratio (A–C) and seizure duration (D–F) between all WT (n = 25) and TPH2-ChR2-YFP mice (n = 24) (A, D), those that survived (WT: n = 10, 4M, 6F; TPH2-ChR2-YFP: n = 14, 5M, 9F) versus died from the MES procedure (WT: n = 15, 10M, 5F; TPH2-ChR2-YFP: n = 10, 5M, 5F) (B, E), and between the female and male mice of each genotype (C, F). Both WT and TPH2-ChR2-YFP mice received pre-ictal optogenetic stimulation. Dots = individual animals. Error bars = SEM. A–F: P ≥ 0.05; A: Mann–Whitney U test; B, C, E, F: one-way ANOVA; D: independent t-test.
Figure 3
Figure 3
Activation of DRD 5-HT neurons reduced MES mortality in TPH2-ChR2-YFP mice, but not WT littermates. (A) Per cent survival of MES following optogenetic stimulation in male and female WT (survived: n = 6; 3M, 3F; died: n = 5; 2M, 3F) and TPH2-ChR2-YFP mice (survived: n = 10, 3M, 7F; died: n = 3, 1M, 2F) with optic fibres implanted within the DRD (*P = 0.0472, Fisher’s exact test). E/F ratio (B, C) and seizure duration (D, E) in WT and TPH2-ChR2-YFP with DRD implants (B, D) and between those that survived (WT: n = 6, 3M, 3F; TPH2-ChR2-YFP: n = 10, 7M, 3F) versus died from the MES procedure (WT: n = 5, 2M, 5F; TPH2-ChR2-YFP: n = 2, 1M, 1F) (C, E). (F) Immunostained coronal section depicting a DRD optic fibre implant. Box indicates location of insets (left, 5-HT cells; middle, ChR2-YFP; right, merge). Dashed lines denote raphe subdivisions. Scale bar = 500 µm, 200 µm. (G) Illustration of midbrain coronal sections demonstrating locations of all DRD hit implants in TPH2-ChR2-YFP and WT mice and whether the animal survived or died from the MES procedure. Dots = individual animals. Error bars = SEM. B–E: P = >0.05; B, D: independent t-test; C, E: one-way ANOVA. DRD, dorsomedial dorsal raphe; DRV, ventral dorsal raphe; DRVL, ventrolateral dorsal raphe; Fibre, optic fibre implant.
Figure 4
Figure 4
Mortality rate was unaffected by activation of non-DRD 5-HT neurons in TPH2-ChR2-YFP mice and WT littermates.  E/F ratio (A, B) and seizure duration (C, D) following optogenetic stimulation and MES in WT and TPH2-ChR2-YFP with non-DRD implants. WT: n = 15, 8M, 6F; TPH2-ChR2-YFP: n = 13, 5M, 8F. (B, D) and between those that survived (WT: n = 5, 2M, 3F; TPH2-ChR2-YFP: n = 5, 2M, 3F) versus died (WT: n = 9, 6M, 3F; TPH2-ChR2-YFP: n = 8, 3M, 5F) from the MES procedure (B, D). (E) Immunostained coronal section depicting an example non-DRD optic fibre implant. Box indicates location of insets (top, 5-HT cells; middle, ChR2-YFP; bottom, merge). Dashed lines denote raphe subdivisions. Scale bar = 250 µm, 100 µm. Dots = individual animals. Error bars = SEM. A–D: P ≥ 0.05; A, C: independent t-test; B, D: one-way ANOVA. Aq, aqueduct; DRD, dorsomedial dorsal raphe; DRV, ventral dorsal raphe; DRVL, ventrolateral dorsal raphe; Fibre, optic fibre implant.
Figure 5
Figure 5
Activation of DRD 5-HT neurons did not affect peri-ictal minute ventilation in TPH2-ChR2-YFP mice. Change in average breathing frequency (breaths/min) (A), tidal volume (B) and minute ventilation (C) during optogenetic stimulation of TPH2-ChR2-YFP DRD hit (n = 7, 2M, 5F) and DRD miss mice (n = 6, 2M, 4F), and WT DRD hit mice (n = 5). Groups consisted of mixed sexes. Dots = individual animals. (*P < 0.0001, Kruskal–Wallis non-parametric test with Dunn’s for multiple comparisons. A: KW = 240.7, B: KW = 1350.0, C: KW = 58.31). Time series of breathing frequency (D), tidal volume (E) and minute ventilation (F) throughout the 300 s of optogenetic stimulation immediately prior to seizure induction (*P < 0.05, mixed-effects model with Sidak’s correction). Dots = group average over 20 s bins. All data were normalized to baseline pre-stimulation values. Error bars = SEM.
Figure 6
Figure 6
Projections were identified from DRD to stress-modulating nuclei, such as the BNST and BLA, but not the HPC. Immunostained coronal sections demonstrating TPH2 expression, fluorogold expression and co-expression within the DRD of a WT mouse following injections of FG into the BNST (A), BLA (B) and HPC (C). Box indicates location of insets (top, FG; middle, TPH2; bottom, merge). (D) Coronal sections from a mouse demonstrating 5-HT2A and CRH RNA transcripts within the anterior-dorsal and anterior-ventral compartments of the BNST. (E) Sagittal section depicting potential DRD to BNST/Amygdala circuitry. ACA, anterior commissure; Aq, aqueduct; BLA, basolateral amygdala; BNST, bed nucleus of the stria terminalis; BNST-AD, anterior-dorsal BNST; BNST-AV, anterior-ventral BNST; DAPI, 4′,6-diamidino-2-phenylindole; DG, dentate gyrus; DRD, dorsomedial dorsal raphe; DRV, dorsal raphe ventral; CA3, cornus ammonis 3; CC, corpus callosum; CP, caudate putamen; CRF, corticotrophin releasing factor; CRFR1 or 2, corticotrophin-releasing factor receptor 1 or 2; GABA, gamma-aminobutyric acid; HPC, hippocampus; Inj, injection; LV, lateral ventricle; PAG, periaqueductal grey. Scale bars: A = 1 mm, 250 µm, 100 µm; B = 2 mm, 500 µm, 100 µm; C = 2.5 mm, 250 µm, 100 µm; D = 250 µm, 100 µm.
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