Nociceptor spontaneous activity is responsible for fragmenting non-rapid eye movement sleep in mouse models of neuropathic pain.

Spontaneous pain, a major complaint of patients with neuropathic pain, has eluded study because there is no reliable marker in either preclinical models or clinical studies. Here, we performed a comprehensive electroencephalogram/electromyogram analysis of sleep in several mouse models of chronic pain: neuropathic (spared nerve injury and chronic constriction injury), inflammatory (Freund's complete adjuvant and carrageenan, plantar incision) and chemical pain (capsaicin). We find that peripheral axonal injury drives fragmentation of sleep by increasing brief arousals from non-rapid eye movement sleep (NREMS) without changing total sleep amount. In contrast to neuropathic pain, inflammatory or chemical pain did not increase brief arousals. NREMS fragmentation was reduced by the analgesics gabapentin and carbamazepine, and it resolved when pain sensitivity returned to normal in a transient neuropathic pain model (sciatic nerve crush). Genetic silencing of peripheral sensory neurons or ablation of CGRP+ neurons in the parabrachial nucleus prevented sleep fragmentation, whereas pharmacological blockade of skin sensory fibers was ineffective, indicating that the neural activity driving the arousals originates ectopically in primary nociceptor neurons and is relayed through the lateral parabrachial nucleus. These findings identify NREMS fragmentation by brief arousals as an effective proxy to measure spontaneous neuropathic pain in mice.

Diet-induced obesity leads to sleep fragmentation independently of the severity of sleep-disordered breathing.

Obesity is associated with sleep-disordered breathing (SDB) and unrefreshing sleep. Residual daytime sleepiness and sleep impairments often persist after SDB treatment in patients with obesity, which suggests an independent effect of obesity on breathing and sleep. However, examining the relationship between sleep architecture and SDB in patients with obesity is complex and can be confounded by multiple factors. The main goal of this study was to examine the relationship between obesity-related changes in sleep architecture and SDB. Sleep recordings were performed in 15 lean C57BL/6J and 17 diet-induced obesity (DIO) mice of the same genetic background. Arousals from sleep and apneas were manually scored. Respiratory arousals were classified as events associated with ≥30% drops in minute ventilation (VE) from baseline. We applied Poincaré analysis of VE during sleep to estimate breathing variability. Obesity augmented the frequency of arousals by 45% and this increase was independent of apneas. Respiratory arousals comprised only 15% of the arousals in both groups of mice. Breathing variability during non-rapid-eye-movment (NREM) sleep was significantly higher in DIO mice, but it was not associated with arousal frequency. Our results suggest that obesity induces sleep fragmentation independently of SDB severity.NEW & NOTEWORTHY Our diet-induced obesity (DIO) model reproduces sleep features of human obesity, including sleep fragmentation, increased apnea frequency, and larger breathing variability. DIO induces sleep fragmentation independently of apnea severity. Sleep fragmentation in DIO mice is mainly attributed to non-respiratory arousals. Increased breathing variability during sleep did not account for the higher arousal frequency in DIO. Our results provide a rationale to examine sleep in patients with obesity even when they are adequately treated for sleep-disordered breathing.

Effect of sleep loss on pain-New conceptual and mechanistic avenues.

Introduction: Sleep disturbances increase pain sensitivity in clinical and preclinical settings, but the precise mechanisms are unknown. This represents a major public health issue because of the growing sleep deficiency epidemic fueled by modern lifestyle. To understand the neural pathways at the intersection between sleep and pain processes, it is critical to determine the precise nature of the sleep disruptions that increase pain and the specific component of the pain response that is targeted. Methods: We performed a review of the literature about sleep disturbances and pain sensitivity in humans and rodents by taking into consideration the targeted sleep stage (REMS, non-NREMS, or both), the amount of sleep lost, and the different types of sleep disruptions (partial or total sleep loss, duration, sleep fragmentation or interruptions), and how these differences might affect distinct components of the pain response. Results: We find that the effects of sleep disturbances on pain are highly conserved among species. The major driver for pain hypersensitivity appears to be the total amount of sleep lost, while REMS loss by itself does not seem to have a direct effect on pain sensitivity. Sleep loss caused by extended wakefulness preferentially increases pain perception, whereas interrupted and limited sleep strongly dysregulates descending controls such as DNIC, especially in women. Discussion: We discuss the possible mechanisms involved, including an increase in inflammatory processes, a loss of nociceptive inhibitory pathways, and a defect in the cognitive processing of noxious input.

Touch and tactile neuropathic pain sensitivity are set by corticospinal projections.

Current models of somatosensory perception emphasize transmission from primary sensory neurons to the spinal cord and on to the brain1-4. Mental influence on perception is largely assumed to occur locally within the brain. Here we investigate whether sensory inflow through the spinal cord undergoes direct top-down control by the cortex. Although the corticospinal tract (CST) is traditionally viewed as a primary motor pathway5, a subset of corticospinal neurons (CSNs) originating in the primary and secondary somatosensory cortex directly innervate the spinal dorsal horn via CST axons. Either reduction in somatosensory CSN activity or transection of the CST in mice selectively impairs behavioural responses to light touch without altering responses to noxious stimuli. Moreover, such CSN manipulation greatly attenuates tactile allodynia in a model of peripheral neuropathic pain. Tactile stimulation activates somatosensory CSNs, and their corticospinal projections facilitate light-touch-evoked activity of cholecystokinin interneurons in the deep dorsal horn. This touch-driven feed-forward spinal-cortical-spinal sensitization loop is important for the recruitment of spinal nociceptive neurons under tactile allodynia. These results reveal direct cortical modulation of normal and pathological tactile sensory processing in the spinal cord and open up opportunities for new treatments for neuropathic pain.

Neuronal-Specific TUBB3 Is Not Required for Normal Neuronal Function but Is Essential for Timely Axon Regeneration.

We generated a knockout mouse for the neuronal-specific ß-tubulin isoform Tubb3 to investigate its role in nervous system formation and maintenance. Tubb3-/- mice have no detectable neurobehavioral or neuropathological deficits, and upregulation of mRNA and protein of the remaining ß-tubulin isotypes results in equivalent total ß-tubulin levels in Tubb3-/- and wild-type mice. Despite similar levels of total ß-tubulin, adult dorsal root ganglia lacking TUBB3 have decreased growth cone microtubule dynamics and a decreased neurite outgrowth rate of 22% in vitro and in vivo. The effect of the 22% slower growth rate is exacerbated for sensory recovery, where fibers must reinnervate the full volume of the skin to recover touch function. Overall, these data reveal that, while TUBB3 is not required for formation of the nervous system, it has a specific role in the rate of peripheral axon regeneration that cannot be replaced by other ß-tubulins.

Time-Resolved Fast Mammalian Behavior Reveals the Complexity of Protective Pain Responses.

Potentially harmful stimuli are detected at the skin by nociceptor sensory neurons that drive rapid protective withdrawal reflexes and pain. We set out to define, at a millisecond timescale, the relationship between the activity of these sensory neurons and the resultant behavioral output. Brief optogenetic activation of cutaneous nociceptors was found to activate only a single action potential in each fiber. This minimal input was used to determine high-speed behavioral responses in freely behaving mice. The localized stimulus generated widespread dynamic repositioning and alerting sub-second behaviors whose nature and timing depended on the context of the animal and its position, activity, and alertness. Our findings show that the primary response to injurious stimuli is not limited, fixed, or localized, but is dynamic, and that it involves recruitment and gating of multiple circuits distributed throughout the central nervous system at a sub-second timescale to effectively both alert to the presence of danger and minimize risk of harm.

Decreased alertness due to sleep loss increases pain sensitivity in mice.

Extended daytime and nighttime activities are major contributors to the growing sleep deficiency epidemic, as is the high prevalence of sleep disorders like insomnia. The consequences of chronic insufficient sleep for health remain uncertain. Sleep quality and duration predict presence of pain the next day in healthy subjects, suggesting that sleep disturbances alone may worsen pain, and experimental sleep deprivation in humans supports this claim. We demonstrate that sleep loss, but not sleep fragmentation, in healthy mice increases sensitivity to noxious stimuli (referred to as 'pain') without general sensory hyper-responsiveness. Moderate daily repeated sleep loss leads to a progressive accumulation of sleep debt and also to exaggerated pain responses, both of which are rescued after restoration of normal sleep. Caffeine and modafinil, two wake-promoting agents that have no analgesic activity in rested mice, immediately normalize pain sensitivity in sleep-deprived animals, without affecting sleep debt. The reversibility of mild sleep-loss-induced pain by wake-promoting agents reveals an unsuspected role for alertness in setting pain sensitivity. Clinically, insufficient or poor-quality sleep may worsen pain and this enhanced pain may be reduced not by analgesics, whose effectiveness is reduced, but by increasing alertness or providing better sleep.

SCOPRISM: a new algorithm for automatic sleep scoring in mice.

BACKGROUND: Scoring of wake-sleep states by trained investigators is a time-consuming task in many sleep experiments. We aimed to validate SCOPRISM, a new open-source algorithm for sleep scoring based on automatic graphical clustering of epoch distribution. METHODS: We recorded sleep and blood pressure signals of 36 orexin-deficient, 7 leptin knock-out, and 43 wild-type control mice in the PRISM laboratory. Additional groups of mice (n=14) and rats (n=6) recorded in independent labs were used to validate the algorithm across laboratories. RESULTS: The overall accuracy, specificity and sensitivity values of SCOPRISM (97%, 95%, and 94%, respectively) on PRISM lab data were similar to those calculated between human scorers (98%, 98%, and 94%, respectively). Using SCOPRISM, we replicated the main sleep and sleep-dependent cardiovascular findings of our previous studies. Finally, the cross-laboratory analyses showed that the SCOPRISM algorithm performed well on mouse and rat data. COMPARISON WITH EXISTING METHODS: SCOPRISM performed similarly or even better than recently reported algorithms. SCOPRISM is a very simple algorithm, extensively (cross)validated and with the possibility to evaluate its efficacy following a quick and easy visual flow chart. CONCLUSIONS: We validated SCOPRISM, a new, automated and open-source algorithm for sleep scoring on a large population of mice, including different mutant strains and on subgroups of mice and rats recorded by independent labs. This algorithm should help accelerate basic research on sleep and integrative physiology in rodents.

Control of arousal by the orexin neurons.

The orexin-producing neurons in the lateral hypothalamus play an essential role in promoting arousal and maintaining wakefulness. These neurons receive a broad variety of signals related to environmental, physiological and emotional stimuli; they project to almost every brain region involved in the regulation of wakefulness; and they fire most strongly during active wakefulness, high motor activation, and sustained attention. This review focuses on the specific neuronal pathways through which the orexin neurons promote wakefulness and maintain high level of arousal, and how recent studies using optogenetic and pharmacogenetic methods have demonstrated that the locus coeruleus, the tuberomammillary nucleus, and the basal forebrain are some of the key sites mediating the arousing actions of orexins.

Altered sleep homeostasis after restraint stress in 5-HTT knock-out male mice: a role for hypocretins.

Restraint stress produces changes in the sleep pattern that are mainly characterized by a delayed increase in rapid eye movement sleep (REMS) amounts. Because the serotonin (5-HT) and the hypocretin (hcrt) systems that regulate REMS are interconnected, we used mutant mice deficient in the 5-HT transporter (5-HTT(-/-)) to examine the role of 5-HT and hcrt neurotransmissions in the sleep response to stress. In contrast to wild-type mice, restraint stress did not induce a delayed increase in REMS amounts in 5-HTT(-/-) mice, indicating impaired sleep homeostasis in mutants. However, pharmacological blockade of the hcrt type 1 receptor (hcrt-R1) before restraint stress restored the REMS increase in 5-HTT(-/-) mice. In line with this finding, 5-HTT(-/-) mutants displayed after restraint stress higher long-lasting activation of hypothalamic preprohcrt neurons than wild-type mice and elevated levels of the hcrt-1 peptide and the hcrt-R1 mRNA in the anterior raphe area. Thus, hypocretinergic neurotransmission was enhanced by stress in 5-HTT(-/-) mice. Furthermore, in 5-HTT(-/-) but not wild-type mice, hypothalamic levels of the 5-HT metabolite 5-hydroxyindole acetic acid significantly increased after restraint stress, indicating a marked enhancement of serotonergic neurotransmission in mutants. Altogether, our data show that increased serotonergic -and in turn hypocretinergic- neurotransmissions exert an inhibitory influence on stress-induced delayed REMS. We propose that the direct interactions between hcrt neurons in the hypothalamus and 5-HT neurons in the anterior raphe nuclei account, at least in part, for the adaptive sleep-wakefulness regulations triggered by acute stress.

Lasting syndrome of depression produced by reduction in serotonin uptake during postnatal development: evidence from sleep, stress, and behavior.

Dysfunction of the serotonin system is implicated in sleep and emotional disorders. To test whether these impairments could arise during development, we studied the impact of early-life, transient versus genetic, permanent alterations of serotonin reuptake on sleep-wakefulness patterns, depression-related behavior, and associated physiological features. Here, we show that female mice treated neonatally with a highly selective serotonin reuptake inhibitor, escitalopram, exhibited signs of depression in the form of sleep anomalies, anhedonia, increased helplessness reversed by chronic antidepressant treatment, enhanced response to acute stress, and increased serotoninergic autoinhibitory feedback. This syndrome was not reproduced by treatment in naive adults but resembled the phenotype of mutant mice lacking the serotonin transporter, except that these exhibited decreased serotonin autoreceptor sensitivity and additional anxiety-like behavior. Thus, alteration of serotonin reuptake during development, whether induced by external or genetic factors, causes a depressive syndrome lasting into adulthood. Such early-life impairments might predispose individuals to sleep and/or mood disorders.

Sleep-stabilizing effects of E-6199, compared to zopiclone, zolpidem and THIP in mice.

UNLABELLED: Gamma aminobutyric acid (GABA)A receptor modulators constitute the majority of clinically used sedative-hypnotics. These compounds have the capacity to initiate and maintain sleep, but decrease REM sleep and delta activity within NREM sleep. In order to avoid such sleep adverse effects, the development of novel compounds remains of interest. STUDY OBJECTIVES: The present study aimed at characterizing the acute effects of a novel putative hypnotic compound, E-6199, compared to zopiclone, zolpidem, and THIP on sleep-wakefulness patterns in mice. We also investigated whether repeated administration (daily injection during 10 days) of E-6199 was associated with tolerance and sleep disturbances at cessation of treatment. MEASUREMENTS AND RESULTS: Polygraphic recordings were performed during 8 h after acute treatment with the various compounds. Under such conditions, E-6199 (5-20 mg/kg i.p.), zopiclone and zolpidem (2-10 mg/kg i.p.), but not THIP (2-10 mg/kg i.p.), exerted a marked sleep-promoting effect. Furthermore, E-6199 specifically increased the duration of NREM and markedly improved sleep continuity by lengthening NREM sleep episodes and reducing short awakenings and microarousal frequency. It also intensified NREM sleep by enhancing the slow wave activity within NREM at wake-NREM transitions. These effects were sustained and became even larger during chronic administration. Finally, abrupt E-6199 withdrawal did not elicit negative sleep effects. CONCLUSIONS: Our findings demonstrate that E-6199 may be an effective hypnotic compound that promotes and improves NREMS, without producing EEG side effects, tolerance or withdrawal phenomena, when administered under chronic conditions.

Early life blockade of 5-hydroxytryptamine 1A receptors normalizes sleep and depression-like behavior in adult knock-out mice lacking the serotonin transporter.

In serotonin transporter knock-out (5-HTT-/-) mice, extracellular serotonin (5-HT) levels are markedly elevated in the brain, and rapid eye movement sleep (REMS) is enhanced compared with wild-type mice. We hypothesized that such sleep impairment at adulthood results from excessive serotonergic tone during early life. Thus, we assessed whether neonatal treatment with drugs capable of limiting the impact of 5-HT on the brain could normalize sleep patterns in 5-HTT-/- mutants. We found that treatments initiated at postnatal day 5 and continued for 2 weeks with the 5-HT synthesis inhibitor para-chlorophenylalanine, or for 4 weeks with the 5-HT(1A) receptor (5-HT(1A)R) antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635), induced total or partial recovery of REMS, respectively, in 5-HTT-/- mutants. Early life treatment with WAY 100635 also reversed the depression-like behavior otherwise observed in these mutants. Possible adaptive changes in 5-HT(1A)R after neonatal treatment with WAY 100635 were investigated by measuring 5-HT(1A) binding sites and 5-HT(1A) mRNA in various REMS- and/or depression-related brain areas, as well as 5-HT(1A)R-mediated hypothermia and inhibition of neuronal firing in the dorsal raphe nucleus. None of these characteristics were modified in parallel with REMS recovery, suggesting that 5-HT(1A)Rs involved in wild-type phenotype rescue in 5-HTT-/- mutants are located in other brain areas or in 5-HT(1A)R-unrelated circuits where they could be transiently expressed during development. The reversal of sleep alterations and depression-like behavior after early life blockade of 5-HT(1A)R in 5-HTT-/- mutants might open new perspectives regarding preventive care of sleep and mood disorders resulting from serotonin transporter impairments during development.