Kctd7 deficiency induces myoclonic seizures associated with Purkinje cell death and microvascular defects.

Mutations in the potassium channel tetramerization domain-containing 7 (KCTD7) gene are associated with a severe neurodegenerative phenotype characterized by childhood onset of progressive and intractable myoclonic seizures accompanied by developmental regression. KCTD7-driven disease is part of a large family of progressive myoclonic epilepsy syndromes displaying a broad spectrum of clinical severity. Animal models of KCTD7-related disease are lacking, and little is known regarding how KCTD7 protein defects lead to epilepsy and cognitive dysfunction. We characterized Kctd7 expression patterns in the mouse brain during development and show that it is selectively enriched in specific regions as the brain matures. We further demonstrate that Kctd7-deficient mice develop seizures and locomotor defects with features similar to those observed in human KCTD7-associated diseases. We also show that Kctd7 is required for Purkinje cell survival in the cerebellum and that selective degeneration of these neurons is accompanied by defects in cerebellar microvascular organization and patterning. Taken together, these results define a new model for KCTD7-associated epilepsy and identify Kctd7 as a modulator of neuron survival and excitability linked to microvascular alterations in vulnerable regions.

X-linked serotonin 2C receptor is associated with a non-canonical pathway for sudden unexpected death in epilepsy.

Sudden Unexpected Death in Epilepsy is a leading cause of epilepsy-related mortality, and the analysis of mouse Sudden Unexpected Death in Epilepsy models is steadily revealing a spectrum of inherited risk phenotypes based on distinct genetic mechanisms. Serotonin (5-HT) signalling enhances post-ictal cardiorespiratory drive and, when elevated in the brain, reduces death following evoked audiogenic brainstem seizures in inbred mouse models. However, no gene in this pathway has yet been linked to a spontaneous epilepsy phenotype, the defining criterion of Sudden Unexpected Death in Epilepsy. Most monogenic models of Sudden Unexpected Death in Epilepsy invoke a failure of inhibitory synaptic drive as a critical pathogenic step. Accordingly, the G protein-coupled, membrane serotonin receptor 5-HT2C inhibits forebrain and brainstem networks by exciting GABAergic interneurons, and deletion of this gene lowers the threshold for lethal evoked audiogenic seizures. Here, we characterize epileptogenesis throughout the lifespan of mice lacking X-linked, 5-HT2C receptors (loxTB Htr2c). We find that loss of Htr2c generates a complex, adult-onset spontaneous epileptic phenotype with a novel progressive hyperexcitability pattern of absences, non-convulsive, and convulsive behavioural seizures culminating in late onset sudden mortality predominantly in male mice. RNAscope localized Htr2c mRNA in subsets of Gad2+ GABAergic neurons in forebrain and brainstem regions. To evaluate the contribution of 5-HT2C receptor-mediated inhibitory drive, we selectively spared their deletion in GAD2+ GABAergic neurons of pan-deleted loxTB Htr2c mice, yet unexpectedly found no amelioration of survival or epileptic phenotype, indicating that expression of 5-HT2C receptors in GAD2+ inhibitory neurons was not sufficient to prevent hyperexcitability and lethal seizures. Analysis of human Sudden Unexpected Death in Epilepsy and epilepsy genetic databases identified an enrichment of HTR2C non-synonymous variants in Sudden Unexpected Death in Epilepsy cases. Interestingly, while early lethality is not reflected in the mouse model, we also identified variants mainly among male Sudden Infant Death Syndrome patients. Our findings validate HTR2C as a novel, sex-linked candidate gene modifying Sudden Unexpected Death in Epilepsy risk, and demonstrate that the complex epilepsy phenotype does not arise solely from 5-HT2C-mediated synaptic disinhibition. These results strengthen the evidence for the serotonin hypothesis of Sudden Unexpected Death in Epilepsy risk in humans, and advance current efforts to develop gene-guided interventions to mitigate premature mortality in epilepsy.

Arx expansion mutation perturbs cortical development by augmenting apoptosis without activating innate immunity in a mouse model of X-linked infantile spasms syndrome.

X-linked infantile spasms syndrome (ISSX) is a clinically devastating developmental epileptic encephalopathy with life-long impact. Arx(GCG)10+7 , a mouse model of the most common triplet-repeat expansion mutation of ARX, exhibits neonatal spasms, electrographic phenotypes and abnormal migration of GABAergic interneuron subtypes. Neonatal presymptomatic treatment with 17ß-estradiol (E2) in Arx(GCG)10+7 reduces spasms and modifies progression of epilepsy. Cortical pathology during this period, a crucial point for clinical intervention in ISSX, has largely been unexplored, and the pathogenic cellular defects that are targeted by early interventions are unknown. In the first postnatal week, we identified a transient wave of elevated apoptosis in Arx(GCG)10+7 mouse cortex that is non-Arx cell autonomous, since mutant Arx-immunoreactive (Arx+) cells are not preferentially impacted by cell death. NeuN+ (also known as Rbfox3) survival was also not impacted, suggesting a vulnerable subpopulation in the immature Arx(GCG)10+7 cortex. Inflammatory processes during this period might explain this transient elevation in apoptosis; however, transcriptomic and immunohistochemical profiling of several markers of inflammation revealed no innate immune activation in Arx(GCG)10+7 cortex. Neither neonatal E2 hormone therapy, nor ACTH(1-24), the frontline clinical therapy for ISSX, diminished the augmented apoptosis in Arx(GCG)10+7 , but both rescued neocortical Arx+ cell density. Since early E2 treatment effectively prevents seizures in this model, enhanced apoptosis does not solely account for the seizure phenotype, but may contribute to other aberrant brain function in ISSX. However, since both hormone therapies, E2 and ACTH(1-24), elevate the density of cortical Arx+-interneurons, their early therapeutic role in other neurological disorders hallmarked by interneuronopathy should be explored.This article has an associated First Person interview with the first author of the paper.

Isoflurane, ketamine-xylazine, and urethane markedly alter breathing even at subtherapeutic doses.

Anesthetics are widely used for animal research on respiratory control in vivo, but their effect on breathing and CO2 chemoreception has not been well characterized in mice, a species now often used for these studies. We previously demonstrated that 1% isoflurane markedly reduces the hypercapnic ventilatory response (HCVR) in adult mice in vivo and masks serotonin [5-hydroxytryptamine (5-HT)] neuron chemosensitivity in vitro. Here we investigated effects of 0.5% isoflurane on breathing in adult mice and also found a large reduction in the HCVR even at this subanesthetic concentration. We then tested the effects on breathing of ketamine-xylazine and urethane, anesthetics widely used in research on breathing. We found that these agents altered baseline breathing and blunted the HCVR at doses within the range typically used experimentally. At lower doses ventilation was decreased, but mice appropriately matched their ventilation to metabolic demands due to a parallel decrease in O2 consumption. Neither ketamine nor urethane decreased chemosensitivity of 5-HT neurons. These results indicate that baseline breathing and/or CO2 chemoreception in mice are decreased by anesthetics widely viewed as not affecting respiratory control, and even at subtherapeutic doses. These effects of anesthetics on breathing may alter the interpretation of studies of respiratory physiology in vivo.NEW & NOTEWORTHY Anesthetics are frequently used in animal research, but their effects on physiological functions in mice have not been well defined. Here we investigated the effects of commonly used anesthetics on breathing in mice. We found that all tested anesthetics significantly reduced the hypercapnic ventilatory response (HCVR), even at subtherapeutic doses. In addition, ketamine-xylazine and urethane anesthesia altered baseline breathing. These data indicate that breathing and the HCVR in mice are highly sensitive to anesthetic modulation.

Isoflurane abolishes spontaneous firing of serotonin neurons and masks their pH/CO2 chemosensitivity.

Serotonin (5-hydroxytryptamine, 5-HT) neurons from the mouse and rat rostral medulla are stimulated by increased CO2 when studied in culture or brain slices. However, the response of 5-HT neurons has been variable when animals are exposed to hypercapnia in vivo. Here we examined whether halogenated inhalational anesthetics, which activate TWIK-related acid-sensitive K(+) (TASK) channels, could mask an effect of CO2 on 5-HT neurons. During in vivo plethysmography in mice, isoflurane (1%) markedly reduced the hypercapnic ventilatory response (HCVR) by 78-96% depending upon mouse strain and ambient temperature. In a perfused rat brain stem preparation, isoflurane (1%) reduced or silenced spontaneous firing of medullary 5-HT neurons in situ and abolished their responses to elevated perfusate Pco2. In dissociated cell cultures, isoflurane (1%) hyperpolarized 5-HT neurons by 6.52 ± 3.94 mV and inhibited spontaneous firing. A subsequent decrease in pH from 7.4 to 7.2 depolarized neurons by 4.07 ± 2.10 mV, but that was insufficient to reach threshold for firing. Depolarizing current restored baseline firing and the firing frequency response to acidosis, indicating that isoflurane did not block the underlying mechanisms mediating chemosensitivity. These results demonstrate that isoflurane masks 5-HT neuron chemosensitivity in vitro and in situ and markedly decreases the HCVR in vivo. The use of this class of anesthetic has a particularly potent inhibitory effect on chemosensitivity of 5-HT neurons.

Mechanisms of sudden unexpected death in epilepsy: the pathway to prevention.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death in patients with refractory epilepsy, with an estimated 35% lifetime risk in this patient population. There is a surprising lack of awareness among patients and physicians of this increased risk of sudden death: in a recent survey, only 33% of Canadian paediatricians who treated patients with epilepsy knew the term SUDEP. Controversy prevails over whether cardiac arrhythmia or respiratory arrest is more important as the primary cause of death. Effective preventive strategies in high-risk patients will rely on definition of the mechanisms that lead from seizures to death. Here, we summarize evidence for the mechanisms that cause cardiac, respiratory and arousal abnormalities during the ictal and postictal period. We highlight potential cellular mechanisms underlying these abnormalities, such as a defect in the serotonergic system, ictal adenosine release, and changes in autonomic output. We discuss genetic mutations that cause Dravet and long QT syndromes, both of which are linked with increased risk of sudden death. We then highlight possible preventive interventions that are likely to decrease SUDEP incidence, including respiratory monitoring in epilepsy monitoring units and overnight supervision. Finally, we discuss treatments, such as selective serotonin reuptake inhibitors, that might be personalized to a specific genetic or pathological defect.

Serotonin neurons and central respiratory chemoreception: where are we now?

Serotonin (5-hydroxytryptamine, 5-HT) neurons are widely considered to play an important role in central respiratory chemoreception. Although many studies in the past decades have supported this hypothesis, there had been concerns about its validity until recently. One recurring claim had been that 5-HT neurons are not consistently sensitive to hypercapnia in vivo. Another belief was that 5-HT neurons do not stimulate breathing; instead, they inhibit or modulate respiratory output. It was also believed by some that 5-HT neuron chemosensitivity is dependent on TASK channels, but mice with genetic deletion of TASK-1 and TASK-3 have a normal hypercapnic ventilatory response. This review explains why these principal arguments against the hypothesis are not supported by existing data. Despite repeated challenges, a large body of evidence now supports the conclusion that at least a subset of 5-HT neurons are central chemoreceptors.

Sudden unexpected death in epilepsy: fatal post-ictal respiratory and arousal mechanisms.

Sudden unexplained death in epilepsy (SUDEP) is the cause of premature death of up to 17% of all patients with epilepsy and as many as 50% with chronic refractory epilepsy. However, SUDEP is not widely recognized to exist. The etiology of SUDEP remains unclear, but growing evidence points to peri-ictal respiratory, cardiac, or autonomic nervous system dysfunction. How seizures affect these systems remains uncertain. Here we focus on respiratory mechanisms believed to underlie SUDEP. We highlight clinical evidence that indicates peri-ictal hypoxemia occurs in a large percentage of patients due to central apnea, and identify the proposed anatomical regions of the brain governing these responses. In addition, we discuss animal models used to study peri-ictal respiratory depression. We highlight the role 5-HT neurons play in respiratory control, chemoreception, and arousal. Finally, we discuss the evidence that 5-HT deficits contribute to SUDEP and sudden infant death syndrome and the striking similarities between the two.