Key pointsr Sudden unexpected death in epilepsy is the leading cause of death in patients with refractory epilepsy.r Respiratory and cardiac impairment induced by a seizure have been identified as possible causes of seizure-related death, but which is more important has been the subject of debate.r Serotonin has been linked to seizure control, but whether it is primarily anti-convulsant or proconvulsant remains controversial.r In this study we induced seizures in mice with a genetic deletion of serotonin neurones and their phenotypically normal littermates while recording EEG, EMG, ECG and breathing, and assessed the effects of seizures on breathing, cardiac activity and survival r Serotonin and serotonin neurones are involved in setting the seizure threshold, regulating seizure severity and preventing mortality, and death in at least one seizure model is due to respiratory arrest, which can be prevented with selective serotonin reuptake inhibitor treatment or 5-HT 2A receptor activation.Abstract Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death in patients with refractory epilepsy. Defects in central control of breathing are important contributors to the pathophysiology of SUDEP, and serotonin (5-HT) system dysfunction may be involved. Here we examined the effect of 5-HT neurone elimination or 5-HT reduction on seizure risk and seizure-induced mortality. Adult Lmx1b f/f/p mice, which lack >99% of 5-HT neurones in the CNS, and littermate controls (Lmx1b f/f ) were subjected to acute seizure induction by maximal electroshock (MES) or pilocarpine, variably including electroencephalography, electrocardiography, plethysmography, mechanical ventilation or pharmacological therapy. Lmx1b f/f/p mice had a lower seizure threshold and increased seizure-induced mortality. Breathing ceased during most seizures without recovery, whereas cardiac activity persisted for up to 9 min before terminal arrest. The mortality rate of mice of both genotypes was reduced by mechanical ventilation during the seizure or 5-HT 2A receptor agonist pretreatment. The selective serotonin reuptake inhibitor citalopram reduced mortality of Lmx1b f/f but not of Lmx1b f/f/p mice. In C57BL/6N mice, reduction of 5-HT synthesis with para-chlorophenylalanine increased MES-induced seizure severity but not mortality. We conclude that 5-HT neurones raise seizure threshold and decrease seizure-related mortality. Death ensued from respiratory failure, followed by terminal asystole. Given that SUDEP often occurs in association with generalised seizures, some mechanisms causing death in our model might be shared with those leading to SUDEP. This model may help determine the relationship between seizures, 5-HT system dysfunction, breathing and death, which may lead to novel ways to prevent SUDEP.

There is a long-standing controversy about the role of serotonin in sleep/wake control, with competing theories that it either promotes sleep or causes arousal. Here, we show that there is a marked increase in wakefulness when all serotonin neurons are genetically deleted in mice hemizygous for ePet1-Cre and homozygous for floxed Lmx1b (Lmx1b f/f/p ). However, this only occurs at cool ambient temperatures and can be explained by a thermoregulatory defect that leads to an increase in motor activity to generate heat. Because some serotonin neurons are stimulated by CO 2 , and serotonin activates thalamocortical networks, we hypothesized that serotonin neurons cause arousal in response to hypercapnia. We found that Lmx1b f/f/p mice completely lacked any arousal response to inhalation of 10% CO 2 (with 21% O 2 in balance N 2 ) but had normal arousal responses to hypoxia, sound, and air puff. We propose that serotonin neurons mediate the potentially life-saving arousal response to hypercapnia. Impairment of this response may contribute to sudden unexpected death in epilepsy, sudden infant death syndrome, and sleep apnea.S erotonin [5-hydroxytryptamine (5-HT)] has long been implicated in the regulation of sleep and wakefulness. However, its specific role remains unclear and controversial (1). 5-HT is considered by some as a sleep-promoting agent, because both pharmacological depletion of 5-HT and chemical lesions of 5-HT neurons lead to insomnia in cats (1-3). However, there are others who consider 5-HT to be a wakefulness promoter (4). Consistent with this, the firing rate of 5-HT neurons is fastest during waking (W), is slower during nonrapid eye movement sleep (NREM) and nearly ceases during rapid eye movement sleep (REM) (5, 6). 5-HT neurons in the dorsal raphé nucleus project to thalamic, cortical, and other structures involved in sleep/wake transitions (7), and 5-HT can convert the firing patterns of thalamic reticular and thalamocortical neurons in slices from a bursting pattern seen in NREM to a tonic single-spiking pattern seen in W (8). 5-HT (along with norepinephrine, histamine, and acetylcholine) is considered by some to be part of the ascending arousal system (AAS), which regulates transitions from sleep to wakefulness (4). However, there is no direct evidence that 5-HT neurons are required for normal arousal, and there is some evidence that they promote NREM (1, 3). Given the complexity of the 5-HT system, it has been difficult to define the roles, either direct or indirect, of 5-HT in different aspects of sleep regulation.In vitro studies have shown that a subset of 5-HT neurons in both the medullary and midbrain raphé increases firing rate in response to a rise in CO 2 or decrease in pH (9). When studied in unanesthetized behaving mammals, similar results have been obtained in vivo by multiple groups (9). 5-HT neurons in both loci are also juxtaposed to large cerebral blood vessels, making them ideally situated to accurately monitor changes in arterial PCO 2 (10, 11). Medullary 5-HT neurons project to ...

Light is a prominent stimulus that synchronizes endogenous circadian rhythmicity to environmental light/ dark cycles. Nocturnal light elevates mRNA of the Period1 (Per1) gene and induces long term state changes, expressed as phase shifts of circadian rhythms. The cellular mechanism for Per1 elevation and light-induced phase advance in the suprachiasmatic nucleus (SCN), a process initiated primarily by glutamatergic neurotransmission from the retinohypothalamic tract, was examined. Glutamate (GLU)-induced phase advances in the rat SCN were blocked by antisense oligodeoxynucleotide (ODN) against Per1 and Ca 2؉ /cAMP response element (CRE)-decoy ODN. CRE-decoy ODN also blocked light-induced phase advances in vivo. Furthermore, the CRE-decoy blocked GLU-induced accumulation of Per1 mRNA. Thus, Ca 2؉ /cAMP response element-binding protein (CREB) and Per1 are integral components of the pathway transducing light-stimulated GLU neurotransmission into phase advance of the circadian clock.Mammalian circadian rhythmicity is generated by endogenous alternations in transcription/translation of putative clock genes within the suprachiasmatic nucleus (SCN) 1 of the basal hypothalamus. As a projection site of the retinohypothalamic tract, the SCN is poised to respond to retinal light information, mediated primarily by glutamatergic (GLU) neurotransmission, to assure time-of-day congruence between the endogenous pacemaker and the external environment. The mechanisms by which the SCN decodes and processes light information are complex and change as the biochemical clock states progress through their 24-h cycle (1). Light resets the clock throughout the night via glutamatergic-N-methyl-D-aspartate receptor-mediated Ca 2ϩ influx, which activates nitric-oxide synthase to liberate nitric oxide (NO) (2). At this point, the light signaling pathway diverges. In the early night, the light-induced state change, which delays subsequent rhythms, proceeds through NO-dependent activation of a neuronal ryanodine receptor. Light-induced state changes in the late night are independent of ryanodine receptor activation, but require activation of protein kinase G (PKG) (3-5). The discovery of several specific genes associated with circadian rhythmicity, including Period (Per) and Timeless (Tim) (for review, see Ref. 6), raises questions regarding the mechanisms that interface nocturnal light signals with the molecular clockwork. Throughout the night, light stimuli sufficient to cause long term state changes, or phase shifts, of circadian rhythms of rodent wheel running correlate with increased phosphorylation of the transcription factor, Ca 2ϩ /cAMP response element-binding protein (CREB) (7, 8), activation of Ca 2ϩ /cAMP response element (CRE)-mediated transcription (9), and a rise in Per1 mRNA (10 -15). This investigation was undertaken to determine whether CRE-mediated activation of Per1 is required for light/GLU-induced phase resetting of the SCN clock. We hypothesized that the GLU-induced phase advance requires activation of CRE and elevation ...

Circadian clocks are complex biochemical systems that cycle with a period of approximately 24 hours. They integrate temporal information regarding phasing of the solar cycle, and adjust their phase so as to synchronize an organism's internal state to the local environmental day and night. Nocturnal light is the dominant regulator of this entrainment. In mammals, information about nocturnal light is transmitted by glutamate released from retinal projections to the circadian clock in the suprachiasmatic nucleus of the hypothalamus. Clock resetting requires the activation of ionotropic glutamate receptors, which mediate Ca2+ influx. The response induced by such activation depends on the clock's temporal state: during early night it delays the clock phase, whereas in late night the clock phase is advanced. To investigate this differential response, we sought signalling elements that contribute solely to phase delay. We analysed intracellular calcium-channel ryanodine receptors, which mediate coupled Ca2+ signalling. Depletion of intracellular Ca2+ stores during early night blocked the effects of glutamate. Activators of ryanodine receptors induced phase resetting only in early night; inhibitors selectively blocked delays induced by light and glutamate. These findings implicate the release of intracellular Ca2+ through ryanodine receptors in the light-induced phase delay of the circadian clock restricted to the early night.