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Objective: SCN8A epileptic encephalopathy is caused predominantly by de novo gain‐of‐function mutations in the voltage‐gated sodium channel Nav1.6. The disorder is characterized by early onset of seizures and developmental delay. Most patients with SCN8A epileptic encephalopathy are refractory to current anti‐seizure medications. Previous studies determining the mechanisms of this disease have focused on neuronal dysfunction as Nav1.6 is expressed by neurons and plays a critical role in controlling neuronal excitability. However, glial dysfunction has been implicated in epilepsy and alterations in glial physiology could contribute to the pathology of SCN8A encephalopathy. In the current study, we examined alterations in astrocyte and microglia physiology in the development of seizures in a mouse model of SCN8A epileptic encephalopathy. Methods: Using immunohistochemistry, we assessed microglia and astrocyte reactivity before and after the onset of spontaneous seizures. Expression of glutamine synthetase and Nav1.6, and Kir4.1 channel currents were assessed in astrocytes in wild‐type (WT) mice and mice carrying the N1768D SCN8A mutation (D/+). Results: Astrocytes in spontaneously seizing D/+ mice become reactive and increase expression of glial fibrillary acidic protein (GFAP), a marker of astrocyte reactivity. These same astrocytes exhibited reduced barium‐sensitive Kir4.1 currents compared to age‐matched WT mice and decreased expression of glutamine synthetase. These alterations were only observed in spontaneously seizing mice and not before the onset of seizures. In contrast, microglial morphology remained unchanged before and after the onset of seizures. Significance: Astrocytes, but not microglia, become reactive only after the onset of spontaneous seizures in a mouse model of SCN8A encephalopathy. Reactive astrocytes have reduced Kir4.1‐mediated currents, which would impair their ability to buffer potassium. Reduced expression of glutamine synthetase would modulate the availability of neurotransmitters to excitatory and inhibitory neurons. These deficits in potassium and glutamate handling by astrocytes could exacerbate seizures in SCN8A epileptic encephalopathy. Targeting astrocytes may provide a new therapeutic approach to seizure suppression. [ABSTRACT FROM AUTHOR]

Objective: Sudden unexpected death in epilepsy (SUDEP) is an unpredictable and devastating comorbidity of epilepsy that is believed to be due to cardiorespiratory failure immediately after generalized convulsive seizures.Methods: We performed cardiorespiratory monitoring of seizure-induced death in mice carrying either a p.Arg1872Trp or p.Asn1768Asp mutation in a single Scn8a allele-mutations identified from patients who died from SUDEP-and of seizure-induced death in pentylenetetrazole-treated wild-type mice.Results: The primary cause of seizure-induced death for all mice was apnea, as (1) apnea began during a seizure and continued for tens of minutes until terminal asystole, and (2) death was prevented by mechanical ventilation. Fatal seizures always included a tonic phase that was coincident with apnea. This tonic phase apnea was not sufficient to produce death, as it also occurred during many nonfatal seizures; however, all seizures that were fatal had tonic phase apnea. We also made the novel observation that continuous tonic diaphragm contraction occurred during tonic phase apnea, which likely contributes to apnea by preventing exhalation, and this was only fatal when breathing did not resume after the tonic phase ended. Finally, recorded seizures from a patient with developmental epileptic encephalopathy with a previously undocumented SCN8A likely pathogenic variant (p.Leu257Val) revealed similarities to those of the mice, namely, an extended tonic phase that was accompanied by apnea.Interpretation: We conclude that apnea coincident with the tonic phase of a seizure, and subsequent failure to resume breathing, are the determining events that cause seizure-induced death in Scn8a mutant mice. ANN NEUROL 2021;89:1023-1035. [ABSTRACT FROM AUTHOR]

Abstract: The retrotrapezoid nucleus (RTN) regulates breathing in a CO2‐ and state‐dependent manner. RTN neurons are glutamatergic and innervate principally the respiratory pattern generator; they regulate multiple aspects of breathing, including active expiration, and maintain breathing automaticity during non‐REM sleep. RTN neurons encode arterial P C O 2/pH via cell‐autonomous and paracrine mechanisms, and via input from other CO2‐responsive neurons. In short, RTN neurons are a pivotal structure for breathing automaticity and arterial P C O 2 homeostasis. The carotid bodies stimulate the respiratory pattern generator directly and indirectly by activating RTN via a neuronal projection originating within the solitary tract nucleus. The indirect pathway operates under normo‐ or hypercapnic conditions; under respiratory alkalosis (e.g. hypoxia) RTN neurons are silent and the excitatory input from the carotid bodies is suppressed. Also, silencing RTN neurons optogenetically quickly triggers a compensatory increase in carotid body activity. Thus, in conscious mammals, breathing is subject to a dual and interdependent feedback regulation by chemoreceptors. Depending on the circumstance, the activity of the carotid bodies and that of RTN vary in the same or the opposite directions, producing additive or countervailing effects on breathing. These interactions are mediated either via changes in blood gases or by brainstem neuronal connections, but their ultimate effect is invariably to minimize arterial P C O 2 fluctuations. We discuss the potential relevance of this dual chemoreceptor feedback to cardiorespiratory abnormalities present in diseases in which the carotid bodies are hyperactive at rest, e.g. essential hypertension, obstructive sleep apnoea and heart failure. [ABSTRACT FROM AUTHOR]

We discuss recent evidence which suggests that the principal central respiratory chemoreceptors are located within the retrotrapezoid nucleus (RTN) and that RTN neurons are directly sensitive to [H+]. RTN neurons are glutamatergic. In vitro, their activation by [H+] requires expression of a proton-activated G protein-coupled receptor (GPR4) and a proton-modulated potassium channel (TASK-2) whose transcripts are undetectable in astrocytes and the rest of the lower brainstem respiratory network. The pH response of RTN neurons is modulated by surrounding astrocytes but genetic deletion of RTN neurons or deletion of both GPR4 and TASK-2 virtually eliminates the central respiratory chemoreflex. Thus, although this reflex is regulated by innumerable brain pathways, it seems to operate predominantly by modulating the discharge rate of RTN neurons, and the activation of RTN neurons by hypercapnia may ultimately derive from their intrinsic pH sensitivity. RTN neurons increase lung ventilation by stimulating multiple aspects of breathing simultaneously. They stimulate breathing about equally during quiet wake and non-rapid eye movement (REM) sleep, and to a lesser degree during REM sleep. The activity of RTN neurons is regulated by inhibitory feedback and by excitatory inputs, notably from the carotid bodies. The latter input operates during normo- or hypercapnia but fails to activate RTN neurons under hypocapnic conditions. RTN inhibition probably limits the degree of hyperventilation produced by hypocapnic hypoxia. RTN neurons are also activated by inputs from serotonergic neurons and hypothalamic neurons. The absence of RTN neurons probably underlies the sleep apnoea and lack of chemoreflex that characterize congenital central hypoventilation syndrome. [ABSTRACT FROM AUTHOR]

The rostral ventrolateral medulla oblongata (RVLM) contains two functionally distinct types of neurons that control and orchestrate cardiovascular and respiratory responses to hypoxia and hypercapnia. One group is composed of the central chemoreceptor neurons of the retrotrapezoid nucleus, which provides a CO2/H+-dependent drive to breathe and serves as an integration centre and a point of convergence of chemosensory information from other central and peripheral sites, including the carotid bodies. The second cluster of RVLM cells forms a population of neurons belonging to the C1 catecholaminergic group that controls sympathetic vasomotor tone in resting conditions and in conditions of hypoxia and hypercapnia. Recent evidence suggests that ATP-mediated purinergic signalling at the level of the RVLM co-ordinates cardiovascular and respiratory responses triggered by hypoxia and hypercapnia by activating retrotrapezoid nucleus and C1 neurons, respectively. The role of ATP-mediated signalling in the RVLM mechanisms of cardiovascular and respiratory activities is the main subject of this short review. [ABSTRACT FROM AUTHOR]

Key points Several brain regions are thought to sense changes in tissue CO2/H+ to regulate breathing (i.e. central chemoreceptors) including the nucleus of the solitary tract (NTS), medullary raphe and retrotrapezoid nucleus (RTN)., Mechanism(s) underlying RTN chemoreception involve direct activation of RTN neurons by H+-mediated inhibition of a resting K+ conductance and indirect activation of RTN neurons by purinergic signalling, most likely from CO2/H+-sensitive astrocytes., Here, we confirm that activation of P2 receptors in the RTN stimulates cardiorespiratory activity, and we show at the cellular and systems level that purinergic signalling is not essential for CO2/H+ sensing in the NTS or medullary raphe., These results support the possibility that purinergic signalling is a unique feature of RTN chemoreception., Abstract Several brain regions are thought to function as important sites of chemoreception including the nucleus of the solitary tract (NTS), medullary raphe and retrotrapezoid nucleus (RTN). In the RTN, mechanisms of chemoreception involve direct H+-mediated activation of chemosensitive neurons and indirect modulation of chemosensitive neurons by purinergic signalling. Evidence suggests that RTN astrocytes are the source of CO2-evoked ATP release. However, it is not clear whether purinergic signalling also influences CO2/H+ responsiveness of other putative chemoreceptors. The goals of this study are to determine if CO2/H+-sensitive neurons in the NTS and medullary raphe respond to ATP, and whether purinergic signalling in these regions influences CO2 responsiveness in vitro and in vivo. In brain slices, cell-attached recordings of membrane potential show that CO2/H+-sensitive NTS neurons are activated by focal ATP application; however, purinergic P2-receptor blockade did not affect their CO2/H+ responsiveness. CO2/H+-sensitive raphe neurons were unaffected by ATP or P2-receptor blockade. In vivo, ATP injection into the NTS increased cardiorespiratory activity; however, injection of a P2-receptor blocker into this region had no effect on baseline breathing or CO2/H+ responsiveness. Injections of ATP or a P2-receptor blocker into the medullary raphe had no effect on cardiorespiratory activity or the chemoreflex. As a positive control we confirmed that ATP injection into the RTN increased breathing and blood pressure by a P2-receptor-dependent mechanism. These results suggest that purinergic signalling is a unique feature of RTN chemoreception. [ABSTRACT FROM AUTHOR]

R2354 --> 763.4 --> Sudden Unexpected Death in Epilepsy (SUDEP) is defined as the sudden, unexpected and unexplained death of a person with epilepsy and accounts for between 8 and 17% of epilepsy‐related deaths, rising to 50% for patients with refractory epilepsy. In a mouse model of SUDEP, we have recently shown that death is due to seizure‐induced respiratory arrest. In addition, apnea is initiated during the tonic phase and tonic respiratory muscle contraction is a possible mechanism of apnea. In the present study, we explore 1) whether tonic activity of the inspiratory rhythm generator in the brainstem and/or 2) upper motor neuron activity in the motor cortex drives ictal apnea. We recorded video, electrocorticogram (ECoG), electrocardiogram (ECG), and breathing via whole body plethysmography in adult mice carrying the human SCN8A encephalopathy mutation p.Asn1768Asp (N1768D; "D/+ mice") during audiogenic seizures. This mutation was identified from a patient that died from SUDEP and D/+ mice have severe convulsive seizures with apnea and many suffer seizure‐induced death. To test the hypothesis that tonic activity from the inspiratory oscillator results in apnea, we implanted fiberoptic ferrules bilaterally into the Bötzinger Complex (BötC) of mice that express Channelrhodopsin2 (ChR2) under the vesicular GABA transporter (VGAT; "VGAT‐ChR2" mice) that were crossed with D/+ mice (Fig. 1A & B). The goal of the experiment is to photostimulate BötC during ictal apnea to inhibit tonic inspiratory activity and produce expiration (Fig. 1C). Seizures were evoked using a 15 kHz pure tone, as we have done before (Fig. 1D). Trains of light pulses (50 ms pulses, 5 mW of 473 nm light) were evoked repetitively during ictal apnea (Fig. 1E & F). However, this did not recover normal breathing rhythm and apnea duration was no different for any photostimulation paradigm versus control (p = 0.7892, F = 0.1747, One‐Way ANOVA; Fig. 1G). Although breathing was not affected during seizures, the effects on baseline breathing were substantial; for example, inspiration was inhibited for a full 10 second photostimulation train (Fig. 1H). To test the necessity of ictal activity from upper motor neurons in the motor cortex are required for generating ictal apnea, we expressed iDREADD receptors in cortical excitatory neurons of D/+ mice and injected CNO i.p. prior to inducing seizures with a 15 kHz pure tone (Fig. 2A). Under control conditions, seizures presented with the usual tonic phase apnea and spike wave discharges (SWDs) in the motor cortex (Fig. 2B). CNO was able to robustly inhibit the SWDs, but the tonic phase and apnea were not affected (Figs. 2C‐E). The effect of 3 mg/kg CNO on ECoG power was significantly greater than the effect on apnea (p = 0.0059, paired t‐test). In sum, we found that the core inspiratory oscillator circuitry in the brainstem is likely bypassed to create tonic inspiratory activity. Furthermore, inhibition of cortical upper motor neurons has no effect on apnea. Thus, our interpretation is that other pools of upper motor neurons must drive the tonic inspiratory activity and apnea. [ABSTRACT FROM AUTHOR]

Key points The retrotrapezoid nucleus (RTN) is an important site of chemoreception, i.e. the mechanism by which the brain regulates breathing in response to changes in tissue CO2/H+., Mechanisms underlying RTN chemoreception appear to involve direct CO2/H+-mediated neuronal activation and indirect neuronal activation by CO2-evoked ATP release (i.e. purinergic signalling) from astrocytes., Here, we show in vitro and in vivo that purinergic signalling in the RTN contributes to ∼30% of RTN chemoreception., Purinergic drive in the RTN involves gap junction hemichannels but not P2Y1 receptors., These results clearly indicate that purinergic signalling contributes to integrated output of the RTN during hypercapnia and thus is an important determinant of respiratory drive. [ABSTRACT FROM AUTHOR]

Central chemoreception is the mechanism by which CO/pH-sensitive neurons (i.e. chemoreceptors) regulate breathing, presumably in response to changes in tissue pH. A region of the brainstem called the retrotrapezoid nucleus (RTN) is thought to be an important site of chemoreception; select neurons (i.e. chemoreceptors) in this region sense changes in CO/H and send excitatory glutamatergic drive to respiratory centres to modulate the depth and frequency of breathing. Purinergic signalling may also contribute to chemoreception; for instance, it was shown in vivo that CO/H facilitates ATP release within the RTN to stimulate breathing, and recent evidence suggests that CO/H-sensitive RTN astrocytes are the source of this purinergic drive to breathe. In this review, we summarize evidence that RTN astrocytes sense changes in CO/H, identify mechanisms that are likely to confer CO/H sensitivity to RTN astrocytes, including inhibition of heteromeric Kir4.1-Kir5.1 channels and activation of a depolarizing inward current generated by the sodium bicarbonate cotransporter, and discuss the extent to which astrocytes contribute to respiratory drive. [ABSTRACT FROM AUTHOR]