Gordon F. Buchanan

A neuronal ryanodine receptor mediates light-induced phase delays of the circadian clock.

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 organisms 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 clocks 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.

Central serotonin neurons are required for arousal to CO2

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 (Lmx1bf/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 CO2, and serotonin activates thalamocortical networks, we hypothesized that serotonin neurons cause arousal in response to hypercapnia. We found that Lmx1bf/f/p mice completely lacked any arousal response to inhalation of 10% CO2 (with 21% O2 in balance N2) 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.

The serotonin axis: Shared mechanisms in seizures, depression, and SUDEP.

There is a growing appreciation that patients with seizures are also affected by a number of comorbid conditions, including an increase in prevalence of depression ( Kanner, 2009 ), sleep apnea ( Chihorek et al., 2007 ), and sudden death ( Ryvlin et al., 2006 ; Tomson et al., 2008 ). The mechanisms responsible for these associations are unclear. Herein we discuss the possibility that underlying pathology in the serotonin (5‐HT) system of patients with epilepsy lowers the threshold for seizures, while also increasing the risk of depression and sudden death. We propose that postictal dysfunction of 5‐HT neurons causes depression of breathing and arousal in some epilepsy patients, and this can lead to sudden unexpected death in epilepsy (SUDEP). We further draw parallels between SUDEP and sudden infant death syndrome (SIDS), which may share pathophysiologic mechanisms, and which have both been linked to defects in the 5‐HT system.

Serotonin neurones have anti-convulsant effects and reduce seizure-induced mortality

Sudden unexpected death in epilepsy is the leading cause of death in patients with refractory epilepsy. 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. Serotonin has been linked to seizure control, but whether it is primarily anti‐convulsant or proconvulsant remains controversial. 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 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‐HT2A receptor activation.

Localization and behaviors in null mice suggest that ASIC1 and ASIC2 modulate responses to aversive stimuli

Acid‐sensing ion channels (ASICs) generate H+‐gated Na+ currents that contribute to neuronal function and animal behavior. Like ASIC1, ASIC2 subunits are expressed in the brain and multimerize with ASIC1 to influence acid‐evoked currents and facilitate ASIC1 localization to dendritic spines. To better understand how ASIC2 contributes to brain function, we localized the protein and tested the behavioral consequences of ASIC2 gene disruption. For comparison, we also localized ASIC1 and studied ASIC1−/− mice. ASIC2 was prominently expressed in areas of high synaptic density, and with a few exceptions, ASIC1 and ASIC2 localization exhibited substantial overlap. Loss of ASIC1 or ASIC2 decreased freezing behavior in contextual and auditory cue fear conditioning assays, in response to predator odor and in response to CO2 inhalation. In addition, loss of ASIC1 or ASIC2 increased activity in a forced swim assay. These data suggest that ASIC2, like ASIC1, plays a key role in determining the defensive response to aversive stimuli. They also raise the question of whether gene variations in both ASIC1 and ASIC2 might affect fear and panic in humans.

Role of chemoreceptors in mediating dyspnea

Dyspnea, or the uncomfortable awareness of respiratory distress, is a common symptom experienced by most people at some point during their lifetime. It is commonly encountered in individuals with pulmonary disease, such as chronic obstructive pulmonary disease (COPD), but can also be seen in healthy individuals after strenuous exercise, at altitude or in response to psychological stress. Dyspnea is a multifactorial sensation involving the brainstem, cortex, and limbic system, as well as mechanoreceptors, irritant receptors and chemoreceptors. Chemoreceptors appear to contribute to the sensation of dyspnea in two ways. They stimulate the respiratory control system in response to hypoxia and/or hypercapnia, and the resultant increase respiratory motor output can be consciously perceived as unpleasant. They also can induce the sensation of dyspnea through an as yet undetermined mechanism-potentially via direct ascending connections to the limbic system and cortex. The goal of this article is to briefly review how changes in blood gases reach conscious awareness and how chemoreceptors are involved in dyspnea.

Role of the M1 receptor in regulating circadian rhythms.

Cholinergic stimuli are potent regulators of the circadian clock in the hypothalamic suprachiasmatic nucleus (SCN). Using a brain slice model, we have found that the SCN clock is subject to muscarinic regulation, a sensitivity expressed only during the night of the clocks 24-h cycle. Pharmacological and signal transduction characteristics are compatible with a response mediated by an M1-like receptor. Molecular manipulation of muscarinic receptors will provide important insights as to the receptor subtype(s) regulating circadian rhythms.

Corticostriatal interactions in the generation of tic-like behaviors after local striatal disinhibition

The pathophysiology of the tics that define Gilles de la Tourette syndrome (TS) is not well understood. Local disinhibition within the striatum has been hypothesized to play a pathogenic role. In support of this, experimental disinhibition by local antagonism of GABA-A receptors within the striatum produces tic-like phenomenology in monkey and rat. We replicated this effect in mice via local picrotoxin infusion into the dorsal striatum. Infusion of picrotoxin into sensorimotor cortex produced similar movements, accompanied by signs of behavioral activation; higher-dose picrotoxin in the cortex produced seizures. Striatal inhibition with local muscimol completely abolished tic-like movements after either striatal or cortical picrotoxin, confirming their dependence on the striatal circuitry; in contrast, cortical muscimol attenuated but did not abolish movements produced by striatal picrotoxin. Striatal glutamate blockade eliminated tic-like movements after striatal picrotoxin, indicating that glutamatergic afferents are critical for their generation. These studies replicate and extend previous work in monkey and rat, providing additional validation for the local disinhibition model of tic generation. Our results reveal a key role for corticostriatal glutamatergic afferents in the generation of tic-like movements in this model.

Impaired Serotonergic Brainstem Function during and after Seizures

Impaired breathing, cardiac function, and arousal during and after seizures are important causes of morbidity and mortality. Previous work suggests that these changes are associated with depressed brainstem function in the ictal and post-ictal periods. Lower brainstem serotonergic systems are postulated to play an important role in cardiorespiratory changes during and after seizures, whereas upper brainstem serotonergic and other systems regulate arousal. However, direct demonstration of seizure-associated neuronal activity changes in brainstem serotonergic regions has been lacking. Here, we performed multiunit and single-unit recordings from medullary raphe and midbrain dorsal raphe nuclei in an established rat seizure model while measuring changes in breathing rate and depth as well as heart rate. Serotonergic neurons were identified by immunohistochemistry. Respiratory rate, tidal volume, and minute ventilation were all significantly decreased during and after seizures in this model. We found that population firing of neurons in the medullary and midbrain raphe on multiunit recordings was significantly decreased during the ictal and post-ictal periods. Single-unit recordings from identified serotonergic neurons in the medullary raphe revealed highly consistently decreased firing during and after seizures. In contrast, firing of midbrain raphe serotonergic neurons was more variable, with a mixture of increases and decreases. The markedly suppressed firing of medullary serotonergic neurons supports their possible role in simultaneously impaired cardiorespiratory function in seizures. Decreased arousal likely arises from depressed population activity of several neuronal pools in the upper brainstem and forebrain. These findings have important implications for preventing morbidity and mortality in people living with epilepsy. SIGNIFICANCE STATEMENT Seizures often cause impaired breathing, cardiac dysfunction, and loss of consciousness. The brainstem and, specifically, brainstem serotonin neurons are thought to play an important role in controlling breathing, cardiac function, and arousal. We used an established rat seizure model to study the overall neuronal activity in the brainstem as well as firing of specific serotonin neurons while measuring cardiorespiratory function. Our results demonstrated overall decreases in brainstem neuronal activity and marked downregulation of lower brainstem serotonin neuronal firing in association with decreased breathing and heart rate during and after seizures. These findings point the way toward new treatments to augment brainstem function and serotonin, aiming to prevent seizure complications and reduce morbidity and mortality in people living with epilepsy.

New light on an old paradox: site-dependent effects of carbachol on circadian rhythms.

Acetylcholine (ACh) was the first neurotransmitter identified as a regulator of mammalian circadian rhythms. When injected in vivo, cholinergics induced biphasic clock resetting at night, similar to nocturnal light exposure. However, the retinohypothalamic tract connecting the eye to the suprachiasmatic nucleus (SCN) uses glutamate (GLU) to transmit light signals. We here resolve this long-standing paradox. Whereas injection of the cholinergic agonist, carbachol, into the mouse ventricular system in vivo induced light-like effects, direct application to the SCN in vitro or in vivo induced a distinct response pattern: phase advance of circadian rhythms throughout the nighttime. These results indicate that a new regulatory pathway, involving an extra-SCN cholinergic synapse accessible via ventricular injection, mediates the light-like cholinergic clock resetting reported previously.