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.

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.

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.

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.

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.

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.

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.

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".

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.

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.

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.

Fluoxetine prevents respiratory arrest without enhancing ventilation in DBA/1 mice.

Sudden unexpected death in epilepsy (SUDEP) is a fatal epileptic event. DBA/1 mice are a relevant animal model for the study of SUDEP, as these mice exhibit seizure-induced respiratory arrest (S-IRA) leading to death, which has been observed in patients with witnessed SUDEP. Fluoxetine, a selective serotonin (5-hydroxytryptamine or 5-HT) reuptake inhibitor (SSRI), reduces S-IRA in DBA/1 mice. Given that DBA/1 mice with S-IRA can be resuscitated using a ventilator, we hypothesized that breathing stimulants can prevent S-IRA and that fluoxetine prevents S-IRA by enhancing ventilation in these mice. Spontaneous respiratory function in anesthetized or awake DBA/1 mice was examined using noninvasive plethysmography before and after administering fluoxetine or breathing stimulants, doxapram, and 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine (PK-THPP). The effects of these drugs on S-IRA in DBA/1 mice were tested. As reported previously, systemic administration of fluoxetine reduced S-IRA in awake DBA/1 mice, but fluoxetine in anesthetized and awake DBA/1 mice did not increase basal ventilation or the ventilatory response to 7% CO2. Both doxapram and PK-THPP increased ventilation in room air and in air+7% CO2 in anesthetized DBA/1 mice. However, neither of the breathing stimulants reduced the incidence of S-IRA. Our studies confirm that fluoxetine reduces S-IRA in DBA/1 mice without enhancing basal ventilation in the absence of seizures. Although breathing stimulants increased ventilation in the absence of seizures, they were ineffective in reducing S-IRA, indicating that drug-induced increases in ventilation are insufficient to compensate for S-IRA in DBA/1 mice.

Serotonin and sudden death: differential effects of serotonergic drugs on seizure-induced respiratory arrest in DBA/1 mice.

In the DBA/1 mouse model of sudden unexpected death in epilepsy (SUDEP), administration of a selective serotonin (5-HT) reuptake inhibitor (SSRI), fluvoxamine, completely suppressed seizure-induced respiratory arrest (S-IRA) at 30 min after administration (i.p.) in a dose-related manner without blocking audiogenic seizures (AGSz), but another SSRI, paroxetine, reduced S-IRA but with a delayed (24 h) onset and significant toxicity. A serotonin-norepinephrine reuptake inhibitor, venlafaxine, reduced S-IRA incidence, but higher doses were ineffective. A selective 5-HT7 agonist, AS-19, was totally ineffective in reducing S-IRA. In developing DBA/1 mice that had not previously experienced AGSz, administration of a nonselective 5-HT antagonist, cyproheptadine, induced a significantly greater incidence of S-IRA than that of saline. This study confirms that certain drugs that enhance the activation of 5-HT receptors are able to prevent S-IRA, but not all serotonergic drugs are equally effective, which may be relevant to the potential use of these drugs for SUDEP prevention. Serotonergic antagonists may be problematic in patients with epilepsy.

Effects of age, sex, and sertraline administration on seizure-induced respiratory arrest in the DBA/1 mouse model of sudden unexpected death in epilepsy (SUDEP).

DBA/1 mice are susceptible to audiogenic seizure-induced respiratory arrest (S-IRA), leading to death, which is a model of human sudden unexpected death in epilepsy (SUDEP). Female DBA/1 mice exhibited 71% susceptibility to S-IRA on the third daily test when seizure testing began at postnatal day (PND) 24-30, which was slightly (>10%) but not significantly lower than males. When initial seizure testing was delayed (to >7 weeks of age), DBA/1 mice of both sexes exhibited significantly reduced S-IRA susceptibility, as compared to mice tested initially at PND 24-30. These sex and age issues had not been previously evaluated and may be important for the future use of this SUDEP model. We also observed that 30 min after administering a selective serotonin reuptake inhibitor (SSRI), sertraline (40, 50, or 75 mg/kg i.p.), a significantly reduced S-IRA incidence in DBA/1 mice occurred without blocking seizures, which may be relevant to SUDEP prevention.

Prevention of seizure-induced sudden death in a chronic SUDEP model by semichronic administration of a selective serotonin reuptake inhibitor.

DBA/1 mice are a chronically susceptible model of sudden unexpected death in epilepsy (SUDEP) that exhibit chronic audiogenic generalized convulsive seizures (GCSs), leading to death from respiratory arrest (RA) if not resuscitated. Serotonin (5-HT) normally enhances respiration in response to elevated CO(2) levels, which occur during GCSs in humans. Selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, increase 5-HT availability. We examined whether fluoxetine can block GCS-induced sudden death in DBA/1 mice. Fluoxetine (15-70 mg/kg ip) was administered acutely with seizure induction at 30minutes or semichronically in five daily doses (20mg/kg/day) with induction after 5 days. Acute fluoxetine (45 or 70 mg/kg) significantly reduced the incidence of RA without blocking seizure susceptibility. Semichronic fluoxetine did not block seizures, but significantly reduced seizure-induced RA, which is consistent with effects of SSRIs on respiration in patients with epilepsy [Bateman LM, Li DS,LiN TC, Seyal M. Epilepsia 2010;51:2211-4]. These findings suggest that treatment with SSRIs should be evaluated for reducing the incidence of SUDEP in patients.

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.

Precipitous induction of audiogenic kindling by activation of adenylyl cyclase in the amygdala.

PURPOSE: Kindling of audiogenic seizure (AGS) involves >or=14 AGS over 1-2 weeks in genetically epilepsy-prone rats (GEPR-9s) and induces gradual seizure duration increases, epileptiform electroencephalography (EEG), and emergence of post tonic clonus (PTC), which are long-lasting. N-methyl-d-aspartate (NMDA)-receptor activation in lateral amygdala (LA) is implicated in AGS kindling initiation. However, the persistence of AGS kindling appears to be dependent on molecular mechanisms initiated by NMDA-receptor activation, which may involve adenylyl cyclase (AC). This study attempted to mimic AGS kindling persistently in nonkindled GEPR-9s by one-time activation of AC in LA. METHODS: The effects of a single focal bilateral microinjection into LA of an AC activator, MPB forskolin {7-Deacetyl-7-[O-(N-methylpiperazino)-gamma-butyryl]-forskolin dihydrochloride} (25-100 pmol/side), on seizure behavior in GEPR-9s were evaluated. RESULTS: One-time bilateral microinjection of MPB forskolin in GEPR-9s precipitously induced an AGS kindling-like effect, which involved significant increases in seizure duration and long-lasting susceptibility to AGS that culminates in PTC. This effect occurred at 24 h after MPB forskolin microinjection and lasted >or=5 weeks. The effect was seen when AGS was initiated at 1 and 12 h after microinjection, but not if AGS was induced only at 24 h, indicating the importance of the temporal proximity of AGS induction to the MPB forskolin microinjection. DISCUSSION: These findings indicate that one-time activation of AC within the NMDA receptor-mediated molecular cascade results in precipitous induction of AGS kindling. These data further suggest that AC activation in the LA may be an important epileptogenic mechanism that subserves the long-lasting persistence of AGS kindling.

DBA/1 mice exhibit chronic susceptibility to audiogenic seizures followed by sudden death associated with respiratory arrest.

One proposed cause of sudden unexpected death in epilepsy (SUDEP) in patients is generalized convulsive seizures with respiratory malfunction. We evaluated DBA/1 mice as a chronic SUDEP model. In DBA/1 mice, audiogenic seizures induced by acoustic stimulation resulted in generalized convulsive seizures followed by respiratory arrest from postnatal day (PND) 21 to 100. The incidence of respiratory arrest susceptibility increased, reaching approximately 90-100% by three to seven daily seizures when testing began on PND 21-30. Respiratory arrest was reversible with resuscitation in approximately 98% of mice, which allows repeated seizure testing. Electrocardiographic activity in DBA/1 mice was detectable for approximately 4-6 minutes after respiratory arrest, indicating that death is likely due to respiratory cessation, as cardiac changes occur later. These findings suggest that DBA/1 mice are a useful chronic SUDEP model. These mice die suddenly from respiratory arrest after generalized convulsive seizures until reaching PND >or=100, allowing testing of chronic preventive treatments for SUDEP.

Calcium channel dysfunction in inferior colliculus neurons of the genetically epilepsy-prone rat.

Voltage-gated calcium (Ca(2+)) channels are thought to play an important role in epileptogenesis and seizure generation. Here, using the whole cell configuration of patch-clamp techniques, we report on the modifications of biophysical and pharmacological properties of high threshold voltage-activated Ca(2+) channel currents in inferior colliculus (IC) neurons of the genetically epilepsy-prone rats (GEPR-3s). Ca(2+) channel currents were measured by depolarizing pulses from a holding potential of - 80 mV using barium (Ba(2+)) as the charge carrier. We found that the current density of high threshold voltage-activated Ca(2+) channels was significantly larger in IC neurons of seizure-naive GEPR-3s compared to control Sprague-Dawley rats, and that seizure episodes further enhanced the current density in the GEPR-3s. The increased current density was reflected by both a - 20 mV shifts in channel activation and a 25% increase in the non-inactivating fraction of channels in seizure-naive GEPR-3s. Such changes were reduced by seizure episodes in the GEPR-3s. Pharmacological analysis of the current density suggests that upregulation of L-, N- and R-type of Ca(2+) channels may contribute to IC neuronal hyperexcitability that leads to seizure susceptibility in the GEPR-3s.

Development of a therapeutics curriculum to enhance knowledge of fourth-year medical students about clinical uses and adverse effects of drugs.

BACKGROUND: Enhanced clinical pharmacology and therapeutics education of medical students is important for improving effective and safe drug therapy. Increased education about pharmacovigilance is needed because serious drug-induced adverse effects are increasing. Fostering the needed scientific approach to prescribing requires knowledge of evidence-based drug therapy, based on understanding clinical trials. Therapeutic agents with novel mechanisms of action are increasingly available, and an unbiased understanding of the risks and benefits of novel agents is also important. These issues can be addressed in clinical pharmacology courses. However, many medical schools lack sufficient clinical pharmacologists to teach such courses. The Southern Illinois University Medical School faculty implemented an Advanced Therapeutics course to address these issues. DESCRIPTION: Development of this course involved defining appropriate content and organizing preclinical pharmacology and clinical faculty into teaching teams. The course was offered to 4th-year medical students and covered clinical trial information, and cutting-edge therapeutic developments. The "ABCs of Pharmacology" is a mental algorithm that was presented in the sophomore year and reintroduced in this course. This algorithm emphasizes pharmacovigilance, which stresses the balance between positive and negative effects of pharmacological agents. General principles of clinical pharmacology and therapeutics were covered by a clinical pharmacologist. Most sessions on specific disease treatment involved an integrated presentation by a preclinical pharmacologist and a clinician with expertise in that topic, often in the context of clinical cases. Other important topics were emphasized, which reinforce individualization of therapy, including pharmacogenomics that may determine idiosyncratic responses. Feedback during and following the course was obtained via questionnaires. EVALUATION: This approach was well received by participating students and graduates. Most students rated this course as a valuable experience. CONCLUSION: This approach appears useful for educating medical students about therapeutics at medical schools that lack sufficient clinical pharmacology faculty to mount such a course.