George B. Richerson

Serotonergic neurons as carbon dioxide sensors that maintain ph homeostasis

Serotonergic neurons in the medulla have recently been shown to be sensors of carbon dioxide and pH. There is compelling evidence that the co-release of serotonin, substance P and thyrotropin-releasing hormone from these neurons stimulates the neural network that controls breathing at numerous sites using many different mechanisms. Serotonergic neurons in the midbrain are also chemosensitive, and might mediate non-respiratory responses to increased carbon dioxide, such as arousal. This role in control of pH homeostasis could provide a neurobiological explanation for the link between changes in the serotonin system and sudden infant death syndrome (SIDS).

Calcium-sensitive potassium channelopathy in human epilepsy and paroxysmal movement disorder

The large conductance calcium-sensitive potassium (BK) channel is widely expressed in many organs and tissues, but its in vivo physiological functions have not been fully defined. Here we report a genetic locus associated with a human syndrome of coexistent generalized epilepsy and paroxysmal dyskinesia on chromosome 10q22 and show that a mutation of the α subunit of the BK channel causes this syndrome. The mutant BK channel had a markedly greater macroscopic current. Single-channel recordings showed an increase in open-channel probability due to a three- to fivefold increase in Ca2+ sensitivity. We propose that enhancement of BK channels in vivo leads to increased excitability by inducing rapid repolarization of action potentials, resulting in generalized epilepsy and paroxysmal dyskinesia by allowing neurons to fire at a faster rate. These results identify a gene that is mutated in generalized epilepsy and paroxysmal dyskinesia and have implications for the pathogenesis of human epilepsy, the neurophysiology of paroxysmal movement disorders and the role of BK channels in neurological disease.

Defects in Breathing and Thermoregulation in Mice with Near-Complete Absence of Central Serotonin Neurons

Serotonergic neurons project widely throughout the CNS and modulate many different brain functions. Particularly important, but controversial, are the contributions of serotonin (5-HT) neurons to respiratory and thermoregulatory control. To better define the roles of 5-HT neurons in breathing and thermoregulation, we took advantage of a unique conditional knock-out mouse in which Lmx1b is genetically deleted in Pet1-expressing cells (Lmx1bf/f/p), resulting in near-complete absence of central 5-HT neurons. Here, we show that the hypercapnic ventilatory response in adult Lmx1bf/f/p mice was decreased by 50% compared with wild-type mice, whereas baseline ventilation and the hypoxic ventilatory response were normal. In addition, Lmx1bf/f/p mice rapidly became hypothermic when exposed to an ambient temperature of 4°C, decreasing core temperature to 30°C within 120 min. This failure of thermoregulation was caused by impaired shivering and nonshivering thermogenesis, whereas thermosensory perception and heat conservation were normal. Finally, intracerebroventricular infusion of 5-HT stimulated baseline ventilation, and rescued the blunted hypercapnic ventilatory response. These data identify a previously unrecognized role of 5-HT neurons in the CO2 chemoreflex, whereby they enhance the response of the rest of the respiratory network to CO2. We conclude that the proper function of the 5-HT system is particularly important under conditions of environmental stress and contributes significantly to the hypercapnic ventilatory response and thermoregulatory cold defense.

The Brainstem and Serotonin in the Sudden Infant Death Syndrome

The sudden infant death syndrome (SIDS) is the sudden death of an infant under one year of age that is typically associated with sleep and that remains unexplained after a complete autopsy and death scene investigation. A leading hypothesis about its pathogenesis is that many cases result from defects in brainstem-mediated protective responses to homeostatic stressors occurring during sleep in a critical developmental period. Here we review the evidence for the brainstem hypothesis in SIDS with a focus upon abnormalities related to the neurotransmitter serotonin in the medulla oblongata, as these are the most robust pathologic findings to date. In this context, we synthesize the human autopsy data with genetic, whole-animal, and cellular data concerning the function and development of the medullary serotonergic system. These emerging data suggest an important underlying mechanism in SIDS that may help lead to identification of infants at risk and specific interventions to prevent death.

Impaired Respiratory and Body Temperature Control Upon Acute Serotonergic Neuron Inhibition

Inducible neuron inhibition reveals essential roles for serotonergic neurons in respiratory and body temperature homeostasis. Physiological homeostasis is essential for organism survival. Highly responsive neuronal networks are involved, but their constituent neurons are just beginning to be resolved. To query brain serotonergic neurons in homeostasis, we used a neuronal silencing tool, mouse RC::FPDi (based on the synthetic G protein–coupled receptor Di), designed for cell type–specific, ligand-inducible, and reversible suppression of action potential firing. In mice harboring Di-expressing serotonergic neurons, administration of the ligand clozapine-N-oxide (CNO) by systemic injection attenuated the chemoreflex that normally increases respiration in response to tissue carbon dioxide (CO2) elevation and acidosis. At the cellular level, CNO suppressed firing rate increases evoked by CO2 acidosis. Body thermoregulation at room temperature was also disrupted after CNO triggering of Di; core temperatures plummeted, then recovered. This work establishes that serotonergic neurons regulate life-sustaining respiratory and thermoregulatory networks, and demonstrates a noninvasive tool for mapping neuron function.

Nonvesicular Inhibitory Neurotransmission via Reversal of the GABA Transporter GAT-1

GABA transporters play an important but poorly understood role in neuronal inhibition. They can reverse, but this is widely thought to occur only under pathological conditions. Here we use a heterologous expression system to show that the reversal potential of GAT-1 under physiologically relevant conditions is near the normal resting potential of neurons and that reversal can occur rapidly enough to release GABA during simulated action potentials. We then use paired recordings from cultured hippocampal neurons and show that GABAergic transmission is not prevented by four methods widely used to block vesicular release. This nonvesicular neurotransmission was potently blocked by GAT-1 antagonists and was enhanced by agents that increase cytosolic [GABA] or [Na(+)] (which would increase GAT-1 reversal). We conclude that GAT-1 regulates tonic inhibition by clamping ambient [GABA] at a level high enough to activate high-affinity GABA(A) receptors and that transporter-mediated GABA release can contribute to phasic inhibition.

Chemosensitivity of rat medullary raphe neurones in primary tissue culture

1 The medullary raphe, within the ventromedial medulla (VMM), contains putative central respiratory chemoreceptors. To study the mechanisms of chemosensitivity in the raphe, rat VMM neurones were maintained in primary dissociated tissue culture, and studied using perforated patch‐clamp recordings. Baseline electrophysiological properties were similar to raphe neurones in brain slices and in vivo. 2 Neurones were exposed to changes in CO2 from 5 % to 3 or 9 % while maintaining a constant [NaHCO3]. Fifty‐one per cent of neurones (n= 210) did not change their firing rate by more than 20 % in response to hypercapnic acidosis. However, 22 % of neurones responded to 9 % CO2 with an increase in firing rate (‘stimulated’), and 27 % of neurones responded with a decrease in firing rate (‘inhibited’). 3 Chemosensitivity has often been considered an all‐or‐none property. Instead, a method was developed to quantify the degree of chemosensitivity. Stimulated neurones had a mean increase in firing rate to 298 ± 215 % of control when pH decreased from 7.40 to 7.19. Inhibited neurones had a mean increase in firing rate to 232 ± 265 % of control when pH increased from 7.38 to 7.57. 4 Neurones were also exposed to isocapnic acidosis. All CO2‐stimulated neurones tested (n= 15) were also stimulated by isocapnic acidosis, and all CO2‐inhibited neurones tested (n= 19) were inhibited by isocapnic acidosis. Neurones with no response to hypercapnic acidosis also had no response to isocapnic acidosis (n= 12). Thus, the effects of CO2 on these neurones were mediated in part via changes in pH. 5 In stimulated neurones, acidosis induced a small increase in the after‐hyperpolarization level of 1.38 ± 1.15 mV per −0.2 pH units, which was dependent on the level of tonic depolarizing current injection. In voltage clamp mode at a holding potential near resting potential, there were small and inconsistent changes in whole‐cell conductance and holding current in both stimulated and inhibited neurones. These results suggest that pH modulates a conductance in stimulated neurones that is activated during repetitive firing, with a reversal potential close to resting potential. 6 The two subtypes of chemosensitive VMM neurones could be distinguished by characteristics other than their response to acidosis. Stimulated neurones had a large multipolar soma, whereas inhibited neurones had a small fusiform soma. Stimulated neurones were more likely than inhibited neurones to fire with the highly regular pattern typical of serotonergic raphe neurones in vivo. 7 Within the medullary raphe, chemosensitivity is a specialization of two distinct neuronal phenotypes. The response of these neurones to physiologically relevant changes in pH is of the magnitude that suggests that this chemosensitivity plays a functional role. Elucidating their mechanisms in vitro may help to define the cellular mechanisms of central chemoreception in vivo.

Raphé Neurons Stimulate Respiratory Circuit Activity by Multiple Mechanisms via Endogenously Released Serotonin and Substance P

Brainstem serotonin (5-HT) neurons modulate activity of many neural circuits in the mammalian brain, but in many cases endogenous mechanisms have not been resolved. Here, we analyzed actions of raphé 5-HT neurons on respiratory network activity including at the level of the pre-Bötzinger complex (pre-BötC) in neonatal rat medullary slices in vitro, and in the more intact nervous system of juvenile rats in arterially perfused brainstem–spinal cord preparations in situ. At basal levels of activity, excitation of the respiratory network via simultaneous release of 5-HT and substance P (SP), acting at 5-HT2A/2C, 5-HT4, and/or neurokinin-1 receptors, was required to maintain inspiratory motor output in both the neonatal and juvenile systems. The midline raphé obscurus contained spontaneously active 5-HT neurons, some of which projected to the pre-BötC and hypoglossal motoneurons, colocalized 5-HT and SP, and received reciprocal excitatory connections from the pre-BötC. Experimentally augmenting raphé obscurus activity increased motor output by simultaneously exciting pre-BötC and motor neurons. Biophysical analyses in vitro demonstrated that 5-HT and SP modulated background cation conductances in pre-BötC and motor neurons, including a nonselective cation leak current that contributed to the resting potential, which explains the neuronal depolarization that augmented motor output. Furthermore, we found that 5-HT, but not SP, can transform the electrophysiological phenotype of some pre-BötC neurons to intrinsic bursters, providing 5-HT with an additional role in promoting rhythm generation. We conclude that raphé 5-HT neurons excite key circuit components required for generation of respiratory motor output.

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.

Chemosensitivity of serotonergic neurons in the rostral ventral medulla.

The medullary raphé contains two subtypes of chemosensitive neuron: one that is stimulated by acidosis and another that is inhibited. Both types of neuron are putative chemoreceptors, proposed to act in opposite ways to modulate respiratory output and other pH sensitive brain functions. In this review, we will discuss the cellular properties of these chemosensitive raphé neurons when studied in vitro using brain slices and primary dissociated cell culture. Quantification of chemosensitivity of raphé neurons indicates that they are highly sensitive to small changes in extracellular pH (pH(o)) between 7.2 and 7.6. Stimulation by acidosis occurs only in the specific phenotypic subset of neurons within the raphé that are serotonergic. These serotonergic neurons also have other properties consistent with a specialized role in chemoreception. Homologous serotonergic neurons are present within the ventrolateral medulla (VLM), and may have contributed to localization of respiratory chemoreception to that region. Chemosensitivity of raphé neurons increases in the postnatal period in rats, in parallel with development of respiratory chemoreception in vivo. An abnormality of serotonergic neurons of the ventral medulla has been identified in victims of sudden infant death syndrome (SIDS). The cellular properties of serotonergic raphé neurons suggest that they play a role in the CNS response to hypercapnia, and that they may contribute to interactions between the sleep/wake cycle and respiratory control.