Enhancing the action of serotonin by three different mechanisms prevents spontaneous seizure-induced mortality in Dravet mice.

OBJECTIVE: Sudden unexpected death in epilepsy (SUDEP) is an underestimated complication of epilepsy. Previous studies have demonstrated that enhancement of serotonergic neurotransmission suppresses seizure-induced sudden death in evoked seizure models. However, it is unclear whether elevated serotonin (5-HT) function will prevent spontaneous seizure-induced mortality (SSIM), which is characteristic of human SUDEP. We examined the effects of 5-HT-enhancing agents that act by three different pharmacological mechanisms on SSIM in Dravet mice, which exhibit a high incidence of SUDEP, modeling human Dravet syndrome. METHODS: Dravet mice of both sexes were evaluated for spontaneous seizure characterization and changes in SSIM incidence induced by agents that enhance 5-HT-mediated neurotransmission. Fluoxetine (a selective 5-HT reuptake inhibitor), fenfluramine (a 5-HT releaser and agonist), SR 57227 (a specific 5-HT3 receptor agonist), or saline (vehicle) was intraperitoneally administered over an 8-day period in Dravet mice, and the effect of these treatments on SSIM was examined. RESULTS: Spontaneous seizures in Dravet mice generally progressed from wild running to tonic seizures with or without SSIM. Fluoxetine at 30 mg/kg, but not at 20 or 5 mg/kg, significantly reduced SSIM compared with the vehicle control. Fenfluramine at 1-10 mg/kg, but not .2 mg/kg, fully protected Dravet mice from SSIM, with all mice surviving. Compared with the vehicle control, SR 57227 at 20 mg/kg, but not at 10 or 5 mg/kg, significantly lowered SSIM. The effect of these drugs on SSIM was independent of sex. SIGNIFICANCE: Our data demonstrate that elevating serotonergic function by fluoxetine, fenfluramine, or SR 57227 significantly reduces or eliminates SSIM in Dravet mice in a sex-independent manner. These findings suggest that deficits in serotonergic neurotransmission likely play an important role in the pathogenesis of SSIM, and fluoxetine and fenfluramine, which are US Food and Drug Administration-approved medications, may potentially prevent SUDEP in at-risk patients.

Response to Singh et al. 2023: It is premature for a unified hypothesis of sudden unexpected death in epilepsy: A great amount of research is still needed to understand the multisystem cascade.

In response to the comments by Singh and colleagues about our recent paper proposing a unified hypothesis of SUDEP, we definitely agree that more research is needed. This research should include studies in other models, including Dravet mice, emphasized by Singh et al. However, we strongly believe the hypothesis is timely, because it is based on the continuing progress on SUDEP-related research on serotonin (5-HT) and adenosine as well as neuroanatomical findings.We propose testing of 5-HT enhancing drugs, neurotoxicity blocking drugs, such as N-methyl-D-aspartate (NMDA) antagonists and periaqueductal gray (PAG) electrical stimulation for SUDEP prevention. There are FDA-approved drugs that enhance the action of 5-HT, including fluoxetine and fenfluramine, which is approved for Dravet syndrome. NMDA antagonists, including memantine and ketamine, are also approved for other disorders. PAG electrical stimulation, which is proposed to activate a suffocation alarm, is also approved to treat other conditions and is known to enhance respiration. Experiments using these methods are currently being carried out in animal studies. If these approaches are validated in SUDEP models, treatments could be evaluated relatively quickly in patients with epilepsy (PWE) who exhibit a biomarker for high SUDEP risk, such as peri-ictal respiratory abnormalities. An example of such a study is the ongoing clinical trial of a selective serotonin reuptake inhibitor in PWE. Although, gene-based therapies may ultimately become treatments of choice to prevent SUDEP, as Singh et al suggested, one or more of the approaches we proposed could become temporizing treatments before gene-based therapies can be available. Establishing genetic treatments would require extensive time for each of the genetic abnormalities associated with SUDEP, and too many PWE are likely to die in the meantime.The temporizing treatments may help to reduce the incidence of SUDEP sooner, which is urgently needed.

A unified hypothesis of SUDEP: Seizure-induced respiratory depression induced by adenosine may lead to SUDEP but can be prevented by autoresuscitation and other restorative respiratory response mechanisms mediated by the action of serotonin on the periaqueductal gray.

Sudden unexpected death in epilepsy (SUDEP) is a major cause of death in people with epilepsy (PWE). Postictal apnea leading to cardiac arrest is the most common sequence of terminal events in witnessed cases of SUDEP, and postconvulsive central apnea has been proposed as a potential biomarker of SUDEP susceptibility. Research in SUDEP animal models has led to the serotonin and adenosine hypotheses of SUDEP. These neurotransmitters influence respiration, seizures, and lethality in animal models of SUDEP, and are implicated in human SUDEP cases. Adenosine released during seizures is proposed to be an important seizure termination mechanism. However, adenosine also depresses respiration, and this effect is mediated, in part, by inhibition of neuronal activity in subcortical structures that modulate respiration, including the periaqueductal gray (PAG). Drugs that enhance the action of adenosine increase postictal death in SUDEP models. Serotonin is also released during seizures, but enhances respiration in response to an elevated carbon dioxide level, which often occurs postictally. This effect of serotonin can potentially compensate, in part, for the adenosine-mediated respiratory depression, acting to facilitate autoresuscitation and other restorative respiratory response mechanisms. A number of drugs that enhance the action of serotonin prevent postictal death in several SUDEP models and reduce postictal respiratory depression in PWE. This effect of serotonergic drugs may be mediated, in part, by actions on brainstem sites that modulate respiration, including the PAG. Enhanced activity in the PAG increases respiration in response to hypoxia and other exigent conditions and can be activated by electrical stimulation. Thus, we propose the unifying hypothesis that seizure-induced adenosine release leads to respiratory depression. This can be reversed by serotonergic action on autoresuscitation and other restorative respiratory responses acting, in part, via the PAG. Therefore, we hypothesize that serotonergic or direct activation of this brainstem site may be a useful approach for SUDEP prevention.

Serotonin 5-HT4 receptors play a critical role in the action of fenfluramine to block seizure-induced sudden death in a mouse model of SUDEP.

RATIONALE: Our previous study showed that the recently approved anticonvulsant drug, fenfluramine, which enhances the release of serotonin (5-hydroxytryptamine, 5-HT) in the brain, prevents seizure-induced respiratory arrest (S-IRA) in the DBA/1 mouse model of sudden unexpected death in epilepsy (SUDEP). The present study examined the role of 5-HT receptor subtypes in mediating the effect of this agent by combined administration of fenfluramine with selective 5-HT receptor antagonists prior to seizure in DBA/1 mice. METHODS: Fenfluramine (15 mg/kg, i.p.) was administered to primed DBA/1 mice, and audiogenic seizure (Sz) was induced 16 h later. Thirty min prior to Sz induction a selective antagonist acting on 5-HT1A, 5-HT2, 5-HT3 5-HT4, 5-HT5A, 5-HT6 or 5-HT7 receptors at a sub-toxic dose was administered, and changes in seizure-induced behaviors were evaluated. Follow-up studies examined the effect of administration of a 5-HT4 receptor agonist, BIMU 8, as well as the effect of co-administration of ineffective doses of fenfluramine and BIMU-8 on Sz behaviors. RESULTS: The 5-HT4 antagonist (GR125487) was the only 5-HT receptor antagonist that was able to reverse the action of fenfluramine to block Sz and S-IRA. Treatment with the 5-HT4 receptor agonist (BIMU-8), or co-administration of ineffective doses of BIMU-8 and fenfluramine significantly reduced the incidence of S-IRA and tonic Sz in DBA/1 mice. The antagonists for 5-HT3, 5-HT5A 5-HT6, and 5-HT7 receptors did not significantly affect the action of fenfluramine. However, the 5-HT1A and the 5-HT2 antagonists enhanced the anticonvulsant effects of fenfluramine. CONCLUSIONS: These findings suggest that the action of fenfluramine to prevent seizure-induced sudden death in DBA/1 mice is mediated primarily by activation of 5-HT4 receptors. These studies are the first to indicate the therapeutic potential of 5-HT4 receptor agonists either alone or in combination with fenfluramine for preventing SUDEP. Enhancement of the anticonvulsant effect of fenfluramine by 5-HT1A and 5-HT2 antagonists may involve presynaptic actions of these antagonists. Thus, the Sz and S-IRA blocking actions of fenfluramine involve complex interactions with several 5-HT receptor subtypes. These data also provide further support for the serotonin hypothesis of SUDEP.

Simultaneous cardiac and respiratory inhibition during seizure precedes death in the DBA/1 audiogenic mouse model of SUDEP.

This study was designed to evaluate cardiac and respiratory dysfunction in a mouse model of sudden unexpected death in epilepsy i.e., SUDEP. We simultaneously monitored respiration via plethysmography and the electrocardiogram via telemetry before, during, and after an audiogenic seizure. DBA/1 mice responded to an acoustic stimulus with one or two cycles of circling and jumping before entering a clonic/tonic seizure. This was followed by death unless the mice were resuscitated by mechanical ventilation using room air. During the initial clonic phase, respiration declined and cardiac rhythm is slowed. By the tonic phase, respiration had ceased, atrial P-waves were absent or dissociated from the QRS complex, and heart rate had decreased from 771±11 to 252±16 bpm. Heart rate further deteriorated terminating in asystole unless the mice were resuscitated at the end of the tonic phase which resulted in abrupt recovery of P-waves and a return to normal sinus rhythm, associated with gasping. Interestingly, P-waves were preserved in the mice treated with methylatropine during the pre-ictal period (to block parasympathetic stimulation) and heart rate remained unchanged through the end of the tonic phase (765±8 vs. 748±21 bpm), but as in control, methylatropine treated mice died from respiratory arrest. These results demonstrate that a clonic/tonic seizure in the DBA/1 mouse results in abrupt and simultaneous respiratory and cardiac depression. Although death clearly results from respiratory arrest, our results suggest that seizure activates two central nervous system pathways in this model-one inhibits respiratory drive, whereas the other inhibits cardiac function via vagal efferents. The abrupt and simultaneous recovery of both respiration and cardiac function with mechanical ventilation within an early post-ictal timeframe shows that the vagal discharge can be rapidly terminated. Understanding the central mechanism associated with the abrupt cardiorespiratory dysfunction and equally abrupt recovery may provide clues for therapeutic targets for SUDEP.

Postictal serotonin levels are associated with peri-ictal apnea.

OBJECTIVE: To determine the relationship between serum serotonin (5-HT) levels, ictal central apnea (ICA), and postconvulsive central apnea (PCCA) in epileptic seizures. METHODS: We prospectively evaluated video EEG, plethysmography, capillary oxygen saturation (SpO2), and ECG for 49 patients (49 seizures) enrolled in a multicenter study of sudden unexpected death in epilepsy (SUDEP). Postictal and interictal venous blood samples were collected after a clinical seizure for measurement of serum 5-HT levels. Seizures were classified according to the International League Against Epilepsy 2017 seizure classification. We analyzed seizures with and without ICA (n = 49) and generalized convulsive seizures (GCS) with and without PCCA (n = 27). RESULTS: Postictal serum 5-HT levels were increased over interictal levels for seizures without ICA (p = 0.01), compared to seizures with ICA (p = 0.21). In patients with GCS without PCCA, serum 5-HT levels were increased postictally compared to interictal levels (p < 0.001), but not in patients with seizures with PCCA (p = 0.22). Postictal minus interictal 5-HT levels also differed between the 2 groups with and without PCCA (p = 0.03). Increased heart rate was accompanied by increased serum 5-HT levels (postictal minus interictal) after seizures without PCCA (p = 0.03) compared to those with PCCA (p = 0.42). CONCLUSIONS: The data suggest that significant seizure-related increases in serum 5-HT levels are associated with a lower incidence of seizure-related breathing dysfunction, and may reflect physiologic changes that confer a protective effect against deleterious phenomena leading to SUDEP. These results need to be confirmed with a larger sample size study.

Emergent Antidepressant Discontinuation Syndrome Misdiagnosed as Delirium in the ICU.

We present a case of serious antidepressant discontinuation syndrome (ADDS) in a 72- year-old woman in the intensive care unit (ICU). Although this syndrome may be mild under ambulatory conditions, ICU patients can experience serious neurocognitive symptoms that are difficult to differentiate from delirium. We report delayed recognition of the ADDS in a patient in the ICU who was initially diagnosed with severe hyperactive delirium. Subsequent to hiatal hernia surgery, the patient was admitted to the ICU for aspiration and was intubated. Due to increasing agitation the patient received high doses of dexmedetomidine, fentanyl, midazolam, and propofol. The patient was treated with high doses of a serotonin norepinephrine reuptake inhibitor (SNRI) antidepressant, duloxetine, for 2 years. However, the antidepressant was not effectively administered postsurgically due to gastroparesis. The signs and symptoms of ADDS can occur cryptogenically if they are partially masked by sedating agents. Due to concern for the discontinuation syndrome and the inability to administer duloxetine via a nasogastric tube, another SNRI, venlafaxine, was administered. Venlafaxine administration allowed unexpectedly prompt dose reduction and then total discontinuation of all sedating agents, allowing liberation from mechanical ventilation and ICU discharge. This case illustrates the importance of avoiding antidepressant discontinuation in the ICU.

Neural activity in the periaqueductal gray and other specific subcortical structures is enhanced when a selective serotonin reuptake inhibitor selectively prevents seizure-induced sudden death in the DBA/1 mouse model of sudden unexpected death in epilepsy.

OBJECTIVE: Sudden unexpected death in epilepsy (SUDEP) is a critical issue in epilepsy, and DBA/1 mice are a useful animal model of this devastating epilepsy sequela. The serotonin hypothesis for SUDEP proposes that modifying serotonergic function significantly alters susceptibility to seizure-induced respiratory arrest (S-IRA). Agents that enhance serotonergic function, including a selective serotonin reuptake inhibitor, fluoxetine, selectively prevent S-IRA in DBA/1 mice. This study examined fluoxetine-induced changes in brain activity using manganese-enhanced magnetic resonance imaging (MEMRI) to reveal sites in the DBA/1 mouse brain where fluoxetine acts to prevent S-IRA. METHODS: DBA/1 mice were subjected to audiogenic seizures (Sz) after saline or fluoxetine (45 mg/kg, intraperitoneal) administration. Control DBA/1 mice received fluoxetine or saline, but Sz were not evoked. A previous MEMRI study established the regions of interest (ROIs) for Sz in the DBA/1 mouse brain, and the present study examined MEMRI differences in the ROIs of these mouse groups. RESULTS: The neural activity in several ROIs was significantly increased in fluoxetine-treated DBA/1 mice that exhibited Sz but not S-IRA when compared to the saline-treated mice that exhibited both Sz and respiratory arrest. These structures included the periaqueductal gray (PAG), amygdala, reticular formation (sensorimotor-limbic network), Kölliker-Fuse nucleus, facial-parafacial group (respiratory network), and pontine raphe. Of these ROIs, only the PAG showed significantly decreased neural activity with saline pretreatment when seizure-induced respiratory arrest occurred as compared to saline treatment without seizure. SIGNIFICANCE: The PAG is known to play an important compensatory role for respiratory distress caused by numerous exigent situations in normal animals. The pattern of fluoxetine-induced activity changes in the present study suggests that PAG may be the most critical target for fluoxetine's action to prevent seizure-induced sudden death. These findings have potential clinical importance, because there is evidence of anomalous serotonergic function and PAG imaging abnormalities in human SUDEP.

Fenfluramine, a serotonin-releasing drug, prevents seizure-induced respiratory arrest and is anticonvulsant in the DBA/1 mouse model of SUDEP.

OBJECTIVE: Prevention of sudden unexpected death in epilepsy (SUDEP) is a critical goal for epilepsy therapy. The DBA/1 mouse model of SUDEP exhibits an elevated susceptibility to seizure-induced death in response to electroconvulsive shock, hyperthermia, convulsant drug, and acoustic stimulation. The serotonin hypothesis of SUDEP is based on findings that treatments which modify serotonergic function significantly alter susceptibility to seizure-induced sudden death in several epilepsy models, including DBA/1 mice. Serotonergic abnormalities have also recently been observed in human SUDEP. Fenfluramine is a drug that enhances serotonin release in the brain. Recent studies have found that the addition of fenfluramine improved seizure control in patients with Dravet syndrome, which has a high incidence of SUDEP. Therefore, we investigated the effects of fenfluramine on seizures and seizure-induced respiratory arrest (S-IRA) in DBA/1 mice. METHODS: The dose and time course of the effects of fenfluramine (i.p.) on audiogenic seizures (Sz) induced by an electric bell in DBA/1 mice were determined. Videos of Sz-induced behaviors were recorded for analysis. Statistical significance (P < 0.05) was evaluated using the chi-square test. RESULTS: Sixteen hours after administration of 15 mg/kg of fenfluramine, a high incidence of selective block of S-IRA susceptibility (P < 0.001) occurred in DBA/1 mice without blocking any convulsive behavior. Thirty minutes after 20-40 mg/kg of fenfluramine, significant reductions of seizure incidence and severity, as well as S-IRA susceptibility occurred, which were long-lasting (≥48 hours). The median effective dose (ED50 ) of fenfluramine for significantly reducing Sz at 30 minutes was 21 mg/kg. SIGNIFICANCE: This study presents the first evidence for the effectiveness of fenfluramine in reducing seizure incidence, severity, and S-IRA susceptibility in a mammalian SUDEP model. The ability of fenfluramine to block S-IRA selectively suggests the potential usefulness of fenfluramine in prophylaxis of SUDEP. These results further confirm and extend the serotonin hypothesis of SUDEP.

Deficient post-ictal cardiorespiratory compensatory mechanisms mediated by the periaqueductal gray may lead to death in a mouse model of SUDEP.

Post-ictal cardiorespiratory failure is implicated as a major cause of sudden unexpected death in epilepsy (SUDEP) in patients. The DBA/1 mouse model of SUDEP is abnormally susceptible to fatal seizure-induced cardiorespiratory failure (S-CRF) induced by convulsant drug, hyperthermia, electroshock, and acoustic stimulation. Clinical and pre-clinical studies have implicated periaqueductal gray (PAG) abnormalities in SUDEP. Recent functional neuroimaging studies observed that S-CRF resulted in selective changes in PAG neuronal activity in DBA/1 mice. The PAG plays a critical compensatory role for respiratory distress caused by numerous physiological challenges in non-epileptic individuals. These observations suggest that abnormalities in PAG-mediated cardiorespiratory modulation may contribute to S-CRF in DBA/1 mice. To evaluate this, electrical stimulation (20 Hz, 20-100 µA, 10 s) was presented in the PAG of anesthetized DBA/1 and C57BL/6 (non-epileptic) control mice, and post-stimulus changes in respiration [inter-breath interval (IBI)] and heart rate variability (HRV) were examined. The post-stimulus period was considered analogous to the post-ictal period when S-CRF occurred in previous DBA/1 mouse studies. PAG stimulation caused significant intensity-related decreases in IBI in both mouse strains. However, this effect was significantly reduced in DBA/1 vis-a-vis C57BL/6 mice. These changes began immediately following cessation of stimulation and remained significant for 10 s. This time period is critical for initiating resuscitation to successfully prevent seizure-induced death in previous DBA/1 mouse experiments. Significant post-stimulus increases in HRV were also seen at ≥60 µA in the PAG in C57BL/6 mice, which were absent in DBA/1 mice. These data along with previous neuroimaging findings suggest that compensatory cardiorespiratory modulation mediated by PAG is deficient, which may be important to the susceptibility of DBA/1 mice to S-CRF. These observations suggest that correcting this deficit pharmacologically or by electrical stimulation may help to prevent S-CRF. These findings further support the potential importance of PAG abnormalities to human SUDEP.

Serum serotonin levels in patients with epileptic seizures.

Profound cardiovascular and/or respiratory dysfunction is part of the terminal cascade in sudden unexpected death in epilepsy (SUDEP). Central control of ventilation is mediated by brainstem rhythm generators, which are influenced by a variety of inputs, many of which use the modulatory neurotransmitter serotonin to mediate important inputs for breathing. The aim of this study was to investigate epileptic seizure-induced changes in serum serotonin levels and whether there are potential implications for SUDEP. Forty-one epileptic patients were pooled into 2 groups based on seizure type as (1) generalized tonic-clonic seizures (GTCS) of genetic generalized epilepsy and focal to bilateral tonic-clonic seizures (FBTCS; n = 19) and (2) focal seizures (n = 26) based on clinical signs using surface video-electroencephalography. Postictal serotonin levels were statistically significantly higher after GTCS and FBTCS compared to interictal levels (P = .002) but not focal seizures (P = .941). The change in serotonin (postictal-interictal) was inversely associated with a shorter duration of tonic phase of generalized seizures. The interictal serotonin level was inversely associated with a shorter period of postictal generalized electroencephalographic suppression. These data suggest that peripheral serum serotonin levels may play a role in seizure features and earlier postseizure recovery; these findings merit further study.

Optogenetic activation of 5-HT neurons in the dorsal raphe suppresses seizure-induced respiratory arrest and produces anticonvulsant effect in the DBA/1 mouse SUDEP model.

Sudden unexpected death in epilepsy (SUDEP) is a devastating epilepsy complication. Seizure-induced respiratory arrest (S-IRA) occurs in many witnessed SUDEP patients and animal models as an initiating event leading to death. Thus, understanding the mechanisms underlying S-IRA will advance the development of preventive strategies against SUDEP. Serotonin (5-HT) is an important modulator for many vital functions, including respiration and arousal, and a deficiency of 5-HT signaling is strongly implicated in S-IRA in animal models, including the DBA/1 mouse. However, the brain structures that contribute to S-IRA remain elusive. We hypothesized that the dorsal raphe (DR), which sends 5-HT projections to the forebrain, is implicated in S-IRA. The present study used optogenetics in the DBA/1 mouse model of SUDEP to selectively activate 5-HT neurons in the DR. Photostimulation of DR 5-HT neurons significantly and reversibly reduced the incidence of S-IRA evoked by acoustic stimulation. Activation of 5-HT neurons in the DR suppressed tonic seizures in most DBA/1 mice without altering the seizure latency and duration of wild running and clonic seizures evoked by acoustic stimulation. This suppressant effect of photostimulation on S-IRA is independent of seizure models, as optogenetic stimulation of DR also reduced S-IRA induced by pentylenetetrazole, a proconvulsant widely used to model human generalized seizures. The S-IRA-suppressing effect of photostimulation was increased by 5-hydroxytryptophan, a chemical precursor for 5-HT synthesis, and was reversed by ondansetron, a specific 5-HT3 receptor antagonist, indicating that reduction of S-IRA by photostimulation of the DR is specifically mediated by enhanced 5-HT neurotransmission. Our findings suggest that deficits in 5-HT neurotransmission in the DR are implicated in S-IRA in DBA/1 mice, and that targeted intervention in the DR is potentially useful for prevention of SUDEP.

Specific subcortical structures are activated during seizure-induced death in a model of sudden unexpected death in epilepsy (SUDEP): A manganese-enhanced magnetic resonance imaging study.

Sudden unexpected death in epilepsy (SUDEP) is a major concern for patients with epilepsy. In most witnessed cases of SUDEP generalized seizures and respiratory failure preceded death, and pre-mortem neuroimaging studies in SUDEP patients observed changes in specific subcortical structures. Our study examined the role of subcortical structures in the DBA/1 mouse model of SUDEP using manganese-enhanced magnetic resonance imaging (MEMRI). These mice exhibit acoustically-evoked generalized seizures leading to seizure-induced respiratory arrest (S-IRA) that results in sudden death unless resuscitation is rapidly instituted. MEMRI data in the DBA/1 mouse brain immediately after acoustically-induced S-IRA were compared to data in C57 (control) mice that were exposed to the same acoustic stimulus that did not trigger seizures. The animals were anesthetized and decapitated immediately after seizure in DBA/1 mice and after an equivalent time in control mice. Comparative T1 weighted MEMRI images were evaluated using a 14T MRI scanner and quantified. We observed significant increases in activity in DBA/1 mice as compared to controls at previously-implicated auditory (superior olivary complex) and sensorimotor-limbic [periaqueductal gray (PAG) and amygdala] networks and also in structures in the respiratory network. The activity at certain raphe nuclei was also increased, suggesting activation of serotonergic mechanisms. These data are consistent with previous findings that enhancing the action of serotonin prevents S-IRA in this SUDEP model. Increased activity in the PAG and the respiratory and raphe nuclei suggest that compensatory mechanisms for apnea may have been activated by S-IRA, but they were not sufficient to prevent death. The present findings indicate that changes induced by S-IRA in specific subcortical structures in DBA/1 mice are consistent with human SUDEP findings. Understanding the changes in brain activity during seizure-induced death in animals may lead to improved approaches directed at prevention of human SUDEP.

Neuropathic pain-induced enhancement of spontaneous and pain-evoked neuronal activity in the periaqueductal gray that is attenuated by gabapentin.

Neuropathic pain is a debilitating pathological condition that is poorly understood. Recent evidence suggests that abnormal central processing occurs during the development of neuropathic pain induced by the cancer chemotherapeutic agent, paclitaxel. Yet, it is unclear what role neurons in supraspinal pain network sites, such as the periaqueductal gray, play in altered behavioral sensitivity seen during chronic pain conditions. To elucidate these mechanisms, we studied the spontaneous and thermally evoked firing patterns of ventrolateral periaqueductal gray (vlPAG) neurons in awake-behaving rats treated with paclitaxel to induce neuropathic pain. In the present study, vlPAG neurons in naive rats exhibited either excitatory, inhibitory, or neutral responses to noxious thermal stimuli, as previously observed. However, after development of behavioral hypersensitivity induced by the chemotherapeutic agent, paclitaxel, vlPAG neurons displayed increased neuronal activity and changes in thermal pain-evoked neuronal activity. This involved elevated levels of spontaneous firing and heightened responsiveness to nonnoxious stimuli (allodynia) as well as noxious thermal stimuli (hyperalgesia) as compared with controls. Furthermore, after paclitaxel treatment, only excitatory neuronal responses were observed for both nonnoxious and noxious thermal stimuli. Systemic administration of gabapentin, a nonopioid analgesic, induced significant dose-dependent decreases in the elevated spontaneous and thermally evoked vlPAG neuronal firing to both nonnoxious and noxious thermal stimuli in rats exhibiting neuropathic pain, but not in naive rats. Thus, these results show a strong correlation between behavioral hypersensitivity to thermal stimuli and increased firing of vlPAG neurons in allodynia and hyperalgesia that occur in this neuropathic pain model.

Abnormalities of serotonergic neurotransmission in animal models of SUDEP.

Sudden unexpected death in epilepsy (SUDEP) is a devastating event, and both DBA/1 and DBA/2 mice have been shown to be relevant animal models for studying SUDEP. DBA mice exhibit seizure-induced respiratory arrest (S-IRA), leading to cardiac arrest and subsequent sudden death after generalized audiogenic seizures (AGSs). This sequence of terminal events is also observed in the majority of witnessed human SUDEP cases. Several pathophysiological mechanisms, including respiratory/cardiac dysfunction, have been proposed to contribute to human SUDEP. Several (but not all) selective serotonin (5-HT) reuptake inhibitors (SSRIs), including fluoxetine, can reversibly block S-IRA, and abnormal expression of 5-HT receptors is found in the brainstem of DBA mice. DBA mice, which do not initially show S-IRA, exhibit S-IRA after treatment with a nonselective 5-HT antagonist. These studies suggest that abnormalities of 5-HT neurotransmission are involved in the pathogenesis of S-IRA in DBA mice. Serotonergic (5-HT) transmission plays an important role in normal respiration, and DBA mice exhibiting S-IRA can be resuscitated using a rodent ventilator. It is important and interesting to know if fluoxetine blocks S-IRA in DBA mice by enhancing respiratory ventilation. To test this, the effects of breathing stimulants, doxapram, and 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine (PK-THPP) were compared with the effects of fluoxetine on S-IRA in DBA/1 mice. Although fluoxetine reduces the incidence of S-IRA in DBA/1 mice, as reported previously, the same dose of fluoxetine fails to enhance baseline respiratory ventilation in the absence of AGSs. Doxapram and PK-THPP augment the baseline ventilation in DBA/1 mice. However, these breathing stimulants are ineffective in preventing S-IRA in DBA/1 mice. These data suggest that fluoxetine blocks S-IRA in DBA/1 mice by cellular/molecular mechanisms other than enhancement of basal ventilation. Future research directions are also discussed. This article is part of a Special Issue entitled "Genetic and Reflex Epilepsies, Audiogenic Seizures and Strains: From Experimental Models to the Clinic".

Serotonergic agents act on 5-HT3 receptors in the brain to block seizure-induced respiratory arrest in the DBA/1 mouse model of SUDEP.

Drugs that enhance the action of serotonin (5-hydroxytrypamine, 5-HT), including several selective serotonin reuptake inhibitors (SSRIs), reduce susceptibility to seizure-induced respiratory arrest (S-IRA) that leads to death in the DBA/1 mouse model of sudden unexpected death in epilepsy (SUDEP). However, it is not clear if specific 5-HT receptors are important in the action of these drugs and whether the brain is the major site of action of these agents in this SUDEP model. The current study examined the actions of agents that affect the 5-HT3 receptor subtype on S-IRA and whether intracerebroventricular (ICV) microinjection of an SSRI would reduce S-IRA susceptibility in DBA/1 mice. The data indicate that systemic administration of SR 57227, a 5-HT3 agonist, was effective in blocking S-IRA in doses that did not block seizures, and the S-IRA blocking effect of the SSRI, fluoxetine, was abolished by coadministration of a 5-HT3 antagonist, ondansetron. Intracerebroventricular administration of fluoxetine in the present study was also able to block S-IRA without blocking seizures. These findings suggest that 5-HT3 receptors play an important role in the block of S-IRA by serotonergic agents, such as SSRIs, which is consistent with the abnormal expression of 5-HT3 receptors in the brainstem of DBA mice observed previously. Taken together, these data indicate that systemically administered serotonergic agents act, at least, in part, in the brain, to reduce S-IRA susceptibility in DBA/1 mice and that 5-HT3 receptors may be important to this effect.

Alcohol withdrawal in epileptic rats - Effects on postictal depression, respiration, and death.

Patients with epilepsy are at risk of sudden unexpected death in epilepsy (SUDEP). The most common series of events in witnessed cases of SUDEP is a generalized convulsive seizure followed by terminal apnea. Risk factors for SUDEP include prolonged postictal depression (PID), as well as alcohol abuse. The present study examined these issues in a genetic epilepsy model that exhibits generalized convulsive audiogenic seizures (AGSz) but rarely exhibits seizure-induced death, the genetically epilepsy-prone rats (GEPR-9s). We evaluated the effect of ethanol withdrawal (ETX) in GEPR-9s on respiration patterns, duration of PID, and the incidence of seizure-induced death. Audiogenic seizures were induced in GEPR-9s and in normal Sprague-Dawley rats, which were subjected to a 4-day binge ethanol protocol, 18-24h after the last ethanol dose. Following the tonic seizures, all GEPR-9s exhibited PID, characterized by loss of the righting reflex and respiratory distress (RD), which were absent during ETX seizures in the normal rats. During ETX, GEPR-9s exhibited significant increases in the duration of PID and RD, compared with vehicle-treated GEPR-9s. A significant increase in the incidence of death following seizure in GEPR-9s subjected to ETX was observed, compared with that in vehicle-treated GEPR-9s and normal rats subjected to ETX. Death in GEPR-9s was preceded by prolonged seizures because, in part, of the emergence of post-tonic generalized clonus. These results indicate that ETX induced significant increases in the duration of PID and RD, which contributed to the greater incidence of mortality in GEPR-9s compared with that in vehicle-treated GEPR-9s and normal rats. These experiments observed an elevated risk of sudden death associated with alcohol withdrawal in a genetic epilepsy model that had previously been identified as a risk factor in human SUDEP.

Susceptibility to seizure-induced sudden death in DBA/2 mice is altered by adenosine.

Sudden unexpected death in epilepsy (SUDEP) is rare but is an important public health burden due to the number of patient years lost. Respiratory dysfunction following generalized convulsive seizure is a common sequence of events in witnessed SUDEP cases. The DBA/2 mouse model of SUDEP exhibits generalized convulsive audiogenic seizures (AGSz), which result in seizure-induced respiratory arrest (S-IRA) in ∼75% of these animals, while the remaining DBA/2 mice exhibit AGSz without S-IRA. SUDEP induction may involve actions of adenosine, which is released during generalized seizures in animals and patients and is known to depress respiration. This study examined the effects of systemic administration of agents that alter the actions of adenosine on the incidence of S-IRA in DBA/2 mice. DBA/2 mice that consistently exhibited AGSz without S-IRA showed a significantly increased incidence of S-IRA following treatment with 5-iodotubercidin, which blocks adenosine metabolism. Treatment of DBA/2 mice that consistently exhibited AGSz followed by S-IRA with a non-selective adenosine antagonist, caffeine, or an A2A adenosine receptor subtype-selective antagonist (SCH 442416) significantly reduced S-IRA incidence. By contrast, an A1 adenosine receptor antagonist (DPCPX) was not effective in reducing S-IRA incidence. These findings suggest that preventative approaches for SUDEP should consider agents that reduce the actions of adenosine.

From unwitnessed fatality to witnessed rescue: Pharmacologic intervention in sudden unexpected death in epilepsy.

The mechanisms of sudden unexpected death in epilepsy (SUDEP) have been difficult to define, as most cases occur unwitnessed, and physiologic recordings have been obtained in only a handful of cases. However, recent data obtained from human cases and experimental studies in animal models have brought us closer to identifying potential mechanisms. Theories of SUDEP should be able to explain how a seizure starting in the forebrain can sometimes lead to changes in brainstem cardiorespiratory control mechanisms. Herein we focus on three major themes of work on the causes of SUDEP. First, evidence is reviewed identifying postictal hypoventilation as a major contributor to the cause of death. Second, data are discussed that brainstem serotonin and adenosine pathways may be involved, as well as how they may contribute. Finally, parallels are drawn between SIDS and SUDEP, and we highlight similarities pointing to the possibility of shared pathophysiology involving combined failure of respiratory and cardiovascular control mechanisms. Knowledge about the causes of SUDEP may lead to potential pharmacologic approaches for prevention. We end by describing how translation of this work may result in future applications to clinical care.

Inhibition of adenosine metabolism induces changes in post-ictal depression, respiration, and mortality in genetically epilepsy prone rats.

A major cause of mortality in epilepsy patients is sudden unexpected death in epilepsy (SUDEP). Post-ictal respiratory dysfunction following generalized convulsive seizures is most commonly observed in witnessed cases of human SUDEP. DBA mouse models of SUDEP are induced by audiogenic seizures (AGSz) and show high incidences of seizure-induced death due to respiratory depression. The relatively low incidence of human SUDEP suggests that it may be useful to examine seizure-associated death in an AGSz model that rarely exhibits sudden death, such as genetically epilepsy-prone rats (GEPR-9s). Adenosine is released extensively during seizures and depresses respiration, which may contribute to seizure-induced death. The present study examined the effects of inhibiting adenosine metabolism on the durations of post-ictal depression (PID) and respiratory distress (RD), changes in blood oxygen saturation (% SpO2), and the incidence of post-seizure mortality in GEPR-9s. Systemic administration of adenosine metabolism inhibitors, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, 30 mg/kg) with 5-Iodotubericidin (5-ITU, 3mg/kg) in GEPR-9s resulted in significant changes in the duration of AGSz-induced PID as compared to vehicle in both genders. These agents also significantly increased the duration of post-seizure RD and significantly decreased the mean% SpO2 after AGSz, as compared to vehicle but only in females. Subsequently, we observed that the incidences of death in both genders 12-48 h post-seizure were significantly greater in drug vs. vehicle treatment. The incidence of death in females was also significantly higher than in males, which is consistent with the elevated seizure sensitivity of female GEPR-9s developmentally. These results support a potentially important role of elevated adenosine levels following generalized seizures in the increased incidence of death in GEPR-9s induced by adenosine metabolism inhibitors. These findings may also be relevant to human SUDEP, in light of the elevated adenosine levels that occur post-ictally in humans and its respiratory depressant actions.