Effects of Skilled Training on Sleep Slow Wave Activity and Cortical Gene Expression in the Rat

Study Objective:

The best characterized marker of sleep homeostasis is the amount of slow wave activity (SWA, 0.5–4 Hz) during NREM sleep. SWA increases as a function of previous waking time and declines during sleep, but the underlying mechanisms remain unclear. We have suggested that SWA homeostasis is linked to synaptic potentiation associated with learning during wakefulness. Indeed, studies in rodents and humans found that SWA increases after manipulations that presumably enhance synaptic strength, but the evidence remains indirect. Here we trained rats in skilled reaching, a task known to elicit long-term potentiation in the trained motor cortex, and immediately after learning measured SWA and cortical protein levels of c-fos and Arc, 2 activity-dependent genes involved in motor learning.

Design:

Intracortical local field potential recordings and training on reaching task.

Setting:

Basic sleep research laboratory.

Patients or Participants:

Long Evans adult male rats.

Interventions:

N/A

Measurements and Results:

SWA increased post-training in the trained cortex (the frontal cortex contralateral to the limb used to learn the task), with smaller or no increase in other cortical areas. This increase was reversible within 1 hour, specific to NREM sleep, and positively correlated with changes in performance during the prior training session, suggesting that it reflects plasticity and not just motor activity. Fos and Arc levels were higher in the trained relative to untrained motor cortex immediately after training, but this asymmetry was no longer present after 1 hour of sleep.

Conclusion:

Learning to reach specifically affects gene expression in the trained motor cortex and, in the same area, increases sleep need as measured by a local change in SWA.

Citation:

Hanlon EC; Faraguna U; Vyazovskiy VV; Tononi G; Cirelli C. Effects of skilled training on sleep slow wave activity and cortical gene expression in the rat. SLEEP 2009;32(6):719-729.     

Microevolution of extensively drug-resistant tuberculosis in Russia

Extensively drug-resistant (XDR) tuberculosis (TB), which is resistant to both first- and second-line antibiotics, is an escalating problem, particularly in the Russian Federation. Molecular fingerprinting of 2348 Mycobacterium tuberculosis isolates collected in Samara Oblast, Russia, revealed that 72% belonged to the Beijing lineage, a genotype associated with enhanced acquisition of drug resistance and increased virulence. Whole-genome sequencing of 34 Samaran isolates, plus 25 isolates representing global M. tuberculosis complex diversity, revealed that Beijing isolates originating in Eastern Europe formed a monophyletic group. Homoplasic polymorphisms within this clade were almost invariably associated with antibiotic resistance, indicating that the evolution of this population is primarily driven by drug therapy. Resistance genotypes showed a strong correlation with drug susceptibility phenotypes. A novel homoplasic mutation in rpoC, found only in isolates carrying a common rpoB rifampicin-resistance mutation, may play a role in fitness compensation. Most multidrug-resistant (MDR) isolates also had mutations in the promoter of a virulence gene, eis, which increase its expression and confer kanamycin resistance. Kanamycin therapy may thus select for mutants with increased virulence, helping preserve bacterial fitness and promoting transmission of drug-resistant TB strains. The East European clade was dominated by two MDR clusters, each disseminated across Samara. Polymorphisms conferring fluoroquinolone resistance were independently acquired multiple times within each cluster, indicating that XDR TB is currently not widely transmitted.     

Sugar-based amphiphilic nanoparticles arrest atherosclerosis in vivo

Atherosclerosis, the build-up of occlusive, lipid-rich plaques in arterial walls, is a focal trigger of chronic coronary, intracranial, and peripheral arterial diseases, which together account for the leading causes of death worldwide. Although the directed treatment of atherosclerotic plaques remains elusive, macrophages are a natural target for new interventions because they are recruited to lipid-rich lesions, actively internalize modified lipids, and convert to foam cells with diseased phenotypes. In this work, we present a nanomedicine platform to counteract plaque development based on two building blocks: first, at the single macrophage level, sugar-based amphiphilic macromolecules (AMs) were designed to competitively block oxidized lipid uptake via scavenger receptors on macrophages; second, for sustained lesion-level intervention, AMs were fabricated into serum-stable core/shell nanoparticles (NPs) to rapidly associate with plaques and inhibit disease progression in vivo. An AM library was designed and fabricated into NP compositions that showed high binding and down-regulation of both MSR1 and CD36 scavenger receptors, yielding minimal accumulation of oxidized lipids. When intravenously administered to a mouse model of cardiovascular disease, these AM NPs showed a pronounced increase in lesion association compared with the control nanoparticles, causing a significant reduction in neointimal hyperplasia, lipid burden, cholesterol clefts, and overall plaque occlusion. Thus, synthetic macromolecules configured as NPs are not only effectively mobilized to lipid-rich lesions but can also be deployed to counteract atheroinflammatory vascular diseases, highlighting the promise of nanomedicines for hyperlipidemic and metabolic syndromes.Cardiovascular disease is responsible for one in every three deaths in the United States (1). Chronically high circulating levels of low-density lipoprotein (LDL) deposit and undergo oxidation (oxLDL) within arterial walls, which consequently stimulates endothelial inflammation and recruitment of circulating monocytes (2). These recruited cells differentiate into macrophages that overexpress scavenger receptors (SR), which internalize oxLDL in an unregulated fashion, propagating the inflammatory cascade and leading to multifocal sites of neo-intimal plaques (3, 4). The prevalent cardiovascular therapeutics, which are focused on lowering circulating levels of LDL, are unable to directly target these developing atherosclerotic lesions (5).To address this unmet need, nanoassemblies have been designed to reach narrow vessels and abrogate the lipid deposition and atheroinflammatory phenomena that catalyze plaque establishment, growth, and ensuing acute or chronic cardiovascular events (6). Reports on recent advances in amphiphilic micelles require release of pharmacologic factors to counteract plaque aggravation and local delivery or the conjugation of targeting ligands to reach areas of atherosclerotic lesions (7–9). The major unresolved challenge to repressing atherogenesis lies in the design of serum-stable systems, targeted to lesion SRs, which can have a long-term impact on plaque dynamics (10, 11).Sugar-based amphiphilic macromolecules (AM) have been conceptualized as fully synthetic constructs that exhibit binding to SRs by mimicking the charge and hydrophobicity of oxidized lipoproteins (12). Using both in vitro and in silico models, AMs were found to competitively bind macrophage SRs, thus inhibiting modified lipid internalization and foam cell formation (13–15). When complexed around hydrophobic core solutes using kinetic fabrication techniques, the AMs form nanoparticles (NPs) that display resistance to unimer release in serum-rich environments (16, 17). Presentation of AMs as NPs established a unique modular design, which could be tuned to alter SR expression in vitro, switching macrophages to an antiatherogenic phenotype (18). Despite these advances, in the absence of a rational materials design strategy, AM therapeutics that elicit superior biodistribution, lesion retention, and lipid burden management in vivo have yet to be realized.In this study, we propose a new paradigm of atherosclerotic therapeutics founded on the rational design, screening, and identification of highly atheroprotective, sugar-based AM compositions integrated within serum-stable NPs (Fig. 1). The central hypothesis is that by targeting atherogenic macrophages, AM NPs will show preferential accumulation at the sites of atherosclerotic plaques and arrest the critical conversion of macrophages to foam cells and the ensuing atheroinflammation. To evaluate this hypothesis, serum-stable, modular NPs were kinetically fabricated with structurally diverse AMs that varied in hydrophobicity, rigidity, charge, and stereochemistry. In contrast to the traditional unitary endpoints of efficacy, a multivariate “atheroprotective” biological feature space was envisioned to guide the rational screening of NPs. Three coupled phenomena that converge to enhance lesion targeting and inhibit macrophage foam cell conversion were considered for the materials design: (i) graded binding to the primary scavenger receptors [macrophage scavenger receptor 1 (MSR1), CD36] to aid lesion accumulation; (ii) competitive inhibition of oxLDL uptake; and (iii) down-regulation of the cell surface expression of MSR1 and CD36. The most atheroprotective NP composition was identified through this multidimensional screening platform, validated via structure–activity correlations and administered to challenge atherosclerotic development in vivo. We report lesion localization and therapeutic efficacy in atherosclerotic ApoE^−/− mice, indicating the potential for synthetic AM NPs as therapeutic agents for plaque management in coronary artery disease.Open in a separate windowFig. 1.The envisioned paradigm to counteract atherosclerotic plaque development aims to repress lipid-scavenging receptors at the level of lesion-based macrophages. Sugar-based AMs were designed to competitively block oxLDL uptake via binding and regulation of SRs on human macrophages. A library of AMs, with systematic variations in charge, stereochemistry, and sugar backbones, was synthesized and kinetically fabricated into serum-stable, core/shell NPs and screened in vitro to identify shell architectures that exhibited maximal atheroprotective potency. To demonstrate lesion-level intervention, the lead NP was administered in an atherosclerosis animal model to challenge lesion development during coronary artery disease.     

Rapid Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates Directly from Clinical Samples

The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures of M. tuberculosis. Here we describe a method utilizing biotinylated RNA baits designed specifically for M. tuberculosis DNA to capture full M. tuberculosis genomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the United Kingdom and Lithuania where a matched culture sample was available, and 2 samples that had failed to grow in culture. M. tuberculosis sequencing data were obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were of high quality (>20× depth and >90% of the genome covered). Results were compared with those of conventional molecular and culture-based methods, and high levels of concordance between phenotypical resistance and predicted resistance based on genotype were observed. High-quality sequence data were obtained from one smear-positive culture-negative case. This study demonstrated for the first time the successful and accurate sequencing of M. tuberculosis genomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalized rather than empirical treatment of drug-resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes.     

Prolonged wakefulness alters neuronal responsiveness to local electrical stimulation of the neocortex in awake rats

Prolonged wakefulness or a lack of sleep lead to cognitive deficits, but little is known about the underlying cellular mechanisms. We recently found that sleep deprivation affects spontaneous neuronal activity in the neocortex of sleeping and awake rats. While it is well known that synaptic responses are modulated by ongoing cortical activity, it remains unclear whether prolonged waking affects responsiveness of cortical neurons to incoming stimuli. By applying local electrical microstimulation to the frontal area of the neocortex, we found that after a 4 h period of waking the initial neuronal response in the contralateral frontal cortex was stronger and more synchronous, and was followed by a more profound inhibition of neuronal spiking as compared with the control condition. These changes in evoked activity suggest increased neuronal excitability and indicate that, after staying awake, cortical neurons become transiently bistable. We propose that some of the detrimental effects of sleep deprivation may be a result of altered neuronal responsiveness to incoming intrinsic and extrinsic inputs.     

Regional pattern of metabolic activation is reflected in the sleep EEG after sleep deprivation combined with unilateral whisker stimulation in mice

Regional differences in EEG slow wave activity (SWA) during sleep after sleep deprivation (SD) may be a consequence of differential metabolic activation of cortical areas. We investigated the relationship between the regional EEG dynamics and 2-deoxyglucose (DG) uptake after SD in mice. Six hours' SD were combined with natural unilateral whisker stimulation in an enriched environment to selectively activate the barrel cortex and motor areas. As expected, an interhemispheric asymmetry of 2-DG uptake was found in the barrel cortex immediately after SD. To test whether sleep contributes to recovery of the asymmetry, the stimulation was followed by either undisturbed sleep or by an additional SD. The asymmetry vanished after recovery sleep but also after the additional period of wakefulness without stimulation. In addition, relative 2-DG uptake in the primary motor cortex and retrosplenial area was significantly higher immediately after the SD than after the additional sleep or wakefulness, whereas no other region differed between the groups. Whisker stimulation elicited a greater increase in EEG SWA during non rapid eye movement sleep in the stimulated hemisphere than in the control hemisphere; this increase lasted for 10 h. Within a hemisphere, the initial increase in SWA was higher in the frontal than in the parietal derivation. We conclude that the regional SWA differences during sleep are use-dependent and may be related to the regional pattern of metabolism during the previous waking episode. However, the regional metabolic recovery is not dependent on sleep, and is not directly reflected in changes in SWA during sleep.     

Dim light in the evening causes coordinated realignment of circadian rhythms,sleep, and short-term memory

Light provides the primary signal for entraining circadian rhythms to the day/night cycle. In addition to rods and cones, the retina contains a small population of photosensitive retinal ganglion cells (pRGCs) expressing the photopigment melanopsin (OPN4). Concerns have been raised that exposure to dim artificial lighting in the evening (DLE) may perturb circadian rhythms and sleep patterns, and OPN4 is presumed to mediate these effects. Here, we examine the effects of 4-h, 20-lux DLE on circadian physiology and behavior in mice and the role of OPN4 in these responses. We show that 2 wk of DLE induces a phase delay of ∼2 to 3 h in mice, comparable to that reported in humans. DLE-induced phase shifts are unaffected in Opn4^−/− mice, indicating that rods and cones are capable of driving these responses in the absence of melanopsin. DLE delays molecular clock rhythms in the heart, liver, adrenal gland, and dorsal hippocampus. It also reverses short-term recognition memory performance, which is associated with changes in preceding sleep history. In addition, DLE modifies patterns of hypothalamic and cortical cFos signals, a molecular correlate of recent neuronal activity. Together, our data show that DLE causes coordinated realignment of circadian rhythms, sleep patterns, and short-term memory process in mice. These effects are particularly relevant as DLE conditions―due to artificial light exposure―are experienced by the majority of the populace on a daily basis.

Light exerts profound effects on physiology and behavior, synchronizing biological rhythms to the light/dark cycle (LD) as well as directly modulating alertness and sleep (1, 2). In mammals, light detected by the eye is the primary time cue synchronizing circadian rhythms of activity and rest, a process termed entrainment. Exposure to light at dawn and dusk plays a key role, adjusting the phase of the master circadian clock in the hypothalamic suprachiasmatic nuclei (SCN) (3–5). Studies on the photoreceptors mediating circadian entrainment led to the identification of a distinct photoreceptor system consisting of a subset of photosensitive retinal ganglion cells (pRGCs) expressing the photopigment melanopsin (OPN4) (6, 7). These cells have a peak sensitivity at ∼480 nm (8, 9), hence differing from the classical visual system, which in humans is most sensitive to light at ∼555 nm, corresponding to the red and green cones of the fovea (10). In addition to modulating image-forming responses via local retinal circuitry, OPN4-expressing pRGC axons project to the SCN and different brain areas, setting the circadian clock and driving nonvisual responses to light (5, 7, 11).How does the mammalian brain adapt to changes in daylength? In humans, exposure to long-day photoperiods delays melatonin onset but advances melatonin offset, hence compressing the internal biological night, relative to short-day photoperiods; this is observed in laboratory studies (12, 13) as well as under naturalistic conditions (14, 15). In laboratory mice, the onset and offset of wheel-running activity change dynamically in response to daylength (16). Long-day photoperiods also cause functional reorganization in the SCN. In vivo multiunit recording in mice shows that 16-h light/8-h dark cycles (16:8 LD) weakens phase clustering of SCN neurons (17). Similarly, PERIOD2::LUCIFERASE bioluminescence signals in the mouse dorsal versus ventral SCN are dissociated after 20:4 LD (18). Weakened intercellular coupling in the SCN reflects a form of plasticity, enhancing adaptability of the circadian system to an increase in daylength (19). In addition, 19:5 LD reduces the number of dopamine neurons in the hypothalamus, increasing behavioral immobility and decreasing exploratory activity in rats (20) and mice (21); seasonal variation in photoperiod is also associated with changes in dopamine levels in the human midbrain (22). In the mouse hippocampus, molecular rhythms such as Per1,2 and Cry1,2 are blunted under 20:4 LD (23); however, the consequence is complex: it improves object and spatial discrimination in the spontaneous recognition memory task but disrupts context discrimination in the fear conditioning task (23).Aberrant lighting at night may lead to disrupted circadian rhythms and sleep, which are associated with many adverse health outcomes, including impaired concentration and performance, mood disturbances, metabolic diseases, cardiovascular and neurological disorders, and cancer (24–26). Numerous studies have characterized the disruptive effects of dim light at night (DLAN) on metabolic and mood-related processes in rodents. In these studies, animals were exposed to dim light for the entire night (27–35). As such, DLAN is highly relevant to conditions in which low-level light exposure continues throughout the night, such as light pollution. However, DLAN is somewhat different from exposure to artificial electrical lighting as experienced by the majority of the populace, who typically experience higher light levels during the day (though lower than natural daylight) but dim light for a short period in the evening (DLE) (14, 15, 36, 37). In humans, DLE exposure delays melatonin rhythms and sleep timing (14, 15, 37) and reduces alertness on the subsequent day (36); these phase-delaying effects of DLE on the circadian system are found under both natural summer (14) and winter photoperiods (15). As such, DLE combines features of both long-day photoperiods and DLAN. While similar to a long-day photoperiod, the extended light phase is of a lower light intensity and may exert different effects in comparison with the higher light levels during the day. Conversely, unlike DLAN, under DLE the evening light exposure only occurs at the start of the biological night when the circadian system is most sensitive to light-induced phase delays (38).Although the effects of long-day photoperiods (12–23) and DLAN (27–35) on circadian physiology and behavior have been extensively studied, the effects of DLE—as produced by artificial light exposure—have received less attention. Here, we investigate the effects of 2 wk of DLE in laboratory mice and the role of OPN4 in mediating these responses. Our choice of dim-light level and duration was based upon human studies conducted in nonlaboratory settings (14, 15, 36, 37), which reported that ∼3 to 4 h of ∼20 to 30 lx artificial lighting exposure increased alertness before bedtime, delayed melatonin timing and sleep onset, and increased sleepiness in the morning. Despite their nocturnality, the mouse phase response curve (PRC) is broadly similar to the human PRC: in both species, light presented during the early night delays circadian rhythms, whereas light presented later at night or early in the morning causes phase advances (5, 38, 39). Our DLE protocol comprises a 12-h light phase at 200 lx, a 4-h evening light period at 20 lx, and an 8-h dark phase. Here, we characterize the effects of 4-h, 20-lux DLE on a) locomotor activity rhythms, b) sleep patterns, c) molecular clocks in peripheral tissues, d) short-term memory process, and e) brain cFos signals.     

Association analysis of the LTA4H gene polymorphisms and pulmonary tuberculosis in 9115 subjects

Immunoregulatory eicosanoids have been implicated in protection from mycobacterial infection in cell and animal models. Recently, a study of the zebrafish embryo demonstrated that mutants of the lta4h gene, which encodes the leukotriene A4 hydrolase (LTA4H) enzyme of the eicosanoid pathway, have hypersusceptibility to Mycobacterium marinum infection. It also reported that heterozygosity at the two single nucleotide polymorphisms rs1978331 and rs2660898 located in introns of the LTA4H gene, a human homologue of lta4h, is associated with protection from pulmonary tuberculosis. To replicate this association we genotyped six LTA4H gene polymorphisms in samples from 3703 pulmonary tuberculosis patients and 5412 healthy controls collected in Russia. We found no evidence of the protective effect of heterozygosity at the polymorphisms rs1978331 and rs2660898 (P = 0.29 and 0.49) and no association of the alleles of any of the six polymorphisms (P = 0.13-0.81). These results suggest that common polymorphisms in the LTA4H gene do not play any major role in susceptibility to clinical pulmonary tuberculosis.     

Epidemiological trends and clinical comparisons of Mycobacterium tuberculosis lineages in Thai TB meningitis

Mycobacterium tuberculosis (MTB) strains were isolated from cerebrospinal fluids collected from individual tuberculous meningitis (TBM) patients from 1996 to 2007 (n = 184) and characterised based on IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, Mycobacterium interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) and large sequence polymorphisms (LSPs). Beijing strains were found to possess the highest transmissibility and proportion in clustered isolates. Beijing strain predomination and stability, at 56% of the genotypic proportion, as well as association with drug resistance in TBM patients, was demonstrated. The proportion of Beijing sublineages revealed that the modern Beijing sublineage showed an increasing trend, whereas the ancestral Beijing sublineage showed a decreasing trend across the three periods. In contrast, there were neither clustered nor multidrug-resistance (MDR) isolates from the Euro-American (EuA) lineage, and the lineage genotypic proportion trend was also decreased. Based on LSPs, only the Beijing, Indo-Oceanic and Euro-American lineages were identified from TBM patients in Thailand. TBM mortality rates were not associated with either drug resistance or significantly different among MTB lineages. This study may support the Beijing genotype strain as most pathogenic causing TBM, with the EuA lineage genotype as the most benign of the strain genotypes tested. The analysis of drug susceptibility also revealed the trend of increasing drug resistance, especially MDR, in TBM patients in Thailand.     

Vitamin D accelerates resolution of inflammatory responses during tuberculosis treatment

Calcidiol, the major circulating metabolite of vitamin D, supports induction of pleiotropic antimicrobial responses in vitro. Vitamin D supplementation elevates circulating calcidiol concentrations, and thus has a potential role in the prevention and treatment of infection. The immunomodulatory effects of administering vitamin D to humans with an infectious disease have not previously been reported. To characterize these effects, we conducted a detailed longitudinal study of circulating and antigen-stimulated immune responses in ninety-five patients receiving antimicrobial therapy for pulmonary tuberculosis who were randomized to receive adjunctive high-dose vitamin D or placebo in a clinical trial, and who fulfilled criteria for per-protocol analysis. Vitamin D supplementation accelerated sputum smear conversion and enhanced treatment-induced resolution of lymphopaenia, monocytosis, hypercytokinaemia, and hyperchemokinaemia. Administration of vitamin D also suppressed antigen-stimulated proinflammatory cytokine responses, but attenuated the suppressive effect of antimicrobial therapy on antigen-stimulated secretion of IL-4, CC chemokine ligand 5, and IFN-α. We demonstrate a previously unappreciated role for vitamin D supplementation in accelerating resolution of inflammatory responses during tuberculosis treatment. Our findings suggest a potential role for adjunctive vitamin D supplementation in the treatment of pulmonary infections to accelerate resolution of inflammatory responses associated with increased risk of mortality.