"Counting sheep PSG": EEGLAB-compatible open-source matlab software for signal processing, visualization, event marking and staging of polysomnographic data.

BACKGROUND: Progress in advancing sleep research employing polysomnography (PSG) has been negatively impacted by the limited availability of widely available, open-source sleep-specific analysis tools. NEW METHOD: Here, we introduce Counting Sheep PSG, an EEGLAB-compatible software for signal processing, visualization, event marking and manual sleep stage scoring of PSG data for MATLAB. RESULTS: Key features include: (1) signal processing tools including bad channel interpolation, down-sampling, re-referencing, filtering, independent component analysis, artifact subspace reconstruction, and power spectral analysis, (2) customizable display of polysomnographic data and hypnogram, (3) event marking mode including manual sleep stage scoring, (4) automatic event detections including movement artifact, sleep spindles, slow waves and eye movements, and (5) export of main descriptive sleep architecture statistics, event statistics and publication-ready hypnogram. COMPARISON WITH EXISTING METHODS: Counting Sheep PSG was built on the foundation created by sleepSMG (https://sleepsmg.sourceforge.net/). The scope and functionalities of the current software have made significant advancements in terms of EEGLAB integration/compatibility, preprocessing, artifact correction, event detection, functionality and ease of use. By comparison, commercial software can be costly and utilize proprietary data formats and algorithms, thereby restricting the ability to distribute and share data and analysis results. CONCLUSIONS: The field of sleep research remains shackled by an industry that resists standardization, prevents interoperability, builds-in planned obsolescence, maintains proprietary black-box data formats and analysis approaches. This presents a major challenge for the field of sleep research. The need for free, open-source software that can read open-format data is essential for scientific advancement to be made in the field.

The EEG correlates and dangerous behavioral consequences of drowsy driving after a single night of mild sleep deprivation.

OBJECTIVE: Here, we investigated the behavioral, cognitive, and electrophysiological impact of mild, acute sleep loss via simultaneously recorded behavioral and electrophysiological measures of vigilance during a "real-world", simulated driving task. METHODS: Participants (N = 34) visited the lab for two testing days where their brain activity and vigilance were simultaneously recorded during a driving simulator task. The driving task lasted approximately 70 mins and consisted of tailgating the lead car at high speed, which braked randomly, requiring participants to react quickly to avoid crashing. The night before testing, participants either slept from 12am-9am (Normally Rested), or 1am-6am (Sleep Restriction). RESULTS: After a single night of mild sleep restriction, sleepiness was increased, participants took longer to brake, missed more braking events, and crashed more often. Brain activity showed more intense alpha burst activity and significant changes in EEG spectral power frequencies related to arousal (e.g., delta, theta, alpha). Importantly, increases in amplitude and number of alpha bursts predicted delays in reaction time when braking. CONCLUSIONS: The findings of this study suggest that a single night of mild sleep loss has significant, negative consequences on driving performance and vigilance, and a clear impact on the physiology of the brain in ways that reflect reduced arousal. SIGNIFICANCE: Understanding neural and cognitive changes associated with sleep loss may lead to important advancements in identifying and preventing potentially dangerous sleep-related lapses in vigilance.

Sleep Enhances Consolidation of Memory Traces for Complex Problem-Solving Skills.

Sleep consolidates memory for procedural motor skills, reflected by sleep-dependent changes in the hippocampal-striatal-cortical network. Other forms of procedural skills require the acquisition of a novel strategy to solve a problem, which recruit overlapping brain regions and specialized areas including the caudate and prefrontal cortex. Sleep preferentially benefits strategy and problem-solving skills over the accompanying motor execution movements. However, it is unclear how acquiring new strategies benefit from sleep. Here, participants performed a task requiring the execution of a sequence of movements to learn a novel cognitive strategy. Participants performed this task while undergoing fMRI before and after an interval of either a full night sleep, a daytime nap, or wakefulness. Participants also performed a motor control task, which precluded the opportunity to learn the strategy. In this way, we subtracted motor execution-related brain activations from activations specific to the strategy. The sleep and nap groups experienced greater behavioral performance improvements compared to the wake group on the strategy-based task. Following sleep, we observed enhanced activation of the caudate in addition to other regions in the hippocampal-striatal-cortical network, compared to wakefulness. This study demonstrates that sleep is a privileged time to enhance newly acquired cognitive strategies needed to solve problems.

EEG and behavioural correlates of mild sleep deprivation and vigilance.

OBJECTIVE: The current study investigated the behavioral, cognitive, and electrophysiological impact of mild (only a few hours) and acute (one night) sleep loss via simultaneously recorded behavioural and physiological measures of vigilance. METHODS: Participants (N = 23) came into the lab for two testing days where their brain activity and vigilance were recorded and assessed. The night before the testing session, participants either slept from 12am to 9am (Normally Rested), or from 1am to 6am (Sleep Restriction). RESULTS: Vigilance was reduced and sleepiness was increased in the Sleep Restricted vs. Normally Rested condition, and this was exacerbated over the course of performing the vigilance task. As well, sleep restriction resulted in more intense alpha bursts. Lastly, EEG spectral power differed in Sleep Restricted vs. Normally Rested conditions as sleep onset progressed, particularly for frequencies reflecting arousal (e.g., delta, alpha, beta). CONCLUSIONS: The findings of this study suggest that only one night of mild sleep loss significantly increases sleepiness and, importantly, reduces vigilance. In addition, this sleep loss has a clear impact on the physiology of the brain in ways that reflect reduced arousal. SIGNIFICANCE: Understanding the neural correlates and cognitive processes associated with loss of sleep may lead to important advancements in identifying and preventing deleterious or potentially dangerous, sleep-related lapses in vigilance.

Sleep preferentially enhances memory for a cognitive strategy but not the implicit motor skills used to acquire it.

Sleep is known to be beneficial to the strengthening of two distinct forms of procedural memory: memory for novel, cognitively simple series of motor movements, and memory for novel, cognitively complex strategies required to solve problems. However, these two types of memory are intertwined, since learning a new cognitive procedural strategy occurs through practice, and thereby also requires the execution of a series of simple motor movements. As a result, it is unclear whether the benefit of sleep results from the enhancement of the cognitive strategy, or the motor skills required to execute the solution. To disentangle the role of sleep in these aspects of procedural memory, we employed two tasks: (1) the Tower of Hanoi (ToH), and, (2) a modified version of the ToH, akin to an implicit Motor Sequence Learning (MSL) task. The MSL task involved the identical series of motor movements as the ToH, but without access to the information necessary to execute the task according to the underlying cognitive procedural strategy. Participants (n = 28) were trained on the 3-disk ToH, then retested on 5-disk versions of both ToH and MSL tasks. Half (n = 15) were trained and immediately tested at 8 PM and retested at 8 AM after a night of sleep. They were retested again at 8 PM after a day of wake (PM-AM-PM condition). The other half (n = 13) were trained and immediately tested at 8 AM, retested at 8 PM after a day of wake, and retested again at 8 AM after a night of sleep (AM-PM-AM condition). ToH performance only improved following a period of sleep. There was no benefit of sleep to implicit MSL. Our results show that sleep, but not wake, allowed individuals to extrapolate what was learned on a simpler 3-disk version of the task to the larger 5-disk problem, which included new elements to which they had not yet been exposed. Here, we isolate the specific role sleep plays for cognitive procedural memory: sleep benefits the cognitive strategy, rather than strengthening implicitly acquired motor sequences required to learn and execute the underlying strategy itself.

Microtubule-associated protein 1A is the fibroblast HMW MAP undergoing mitogen-stimulated serine phosphorylation.

A variety of antibodies to microtubule-associated protein (MAP) have been used to demonstrate that phosphorylation of a 350 kDa microtubule-associated protein is stimulated 2-3 fold by epidermal growth factor or serum in quiescent 3T3-L1 fibroblasts and by insulin in 3T3-L1 adipocytes. Phosphorylation occurs on serine residues, and is maximal by 15-20 min. The phosphoprotein has been identified as MAP1A by specific immunoprecipitation with a well-characterized monoclonal antibody.

Characterization of insulin-stimulated microtubule-associated protein kinase. Rapid isolation and stabilization of a novel serine/threonine kinase from 3T3-L1 cells.

A protein kinase, termed microtubule-associated protein (MAP) kinase, which phosphorylates microtubule-associated protein 2 (MAP-2) in vitro and is stimulated 1.5-3-fold in extracts from insulin-treated 3T3-L1 cells has been identified (Ray, L.B., and Sturgill, T.W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1502-1506). Here, we describe chromatographic properties of MAP kinase and provide biochemical characterization of the partially purified enzyme. Isolation of the enzyme is facilitated by its unusually high affinity for hydrophobic interaction chromatography matrices. The molecular weight of the partially purified enzyme was determined to be 35,000 by gel filtration chromatography and 37,000 by glycerol gradient centrifugation. MAP kinase activity of chromatographic fractions correlated precisely with the presence of a 40-kDa phosphoprotein detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MAP kinase has a Km of 7 microM for ATP and does not utilize GTP. Acetyl-CoA carboxylase, ATP citrate-lyase, casein, histones, phosvitin, protamine, and ribosomal protein S6 were all poor substrates relative to MAP-2. The enzyme is inhibited by fluoride and beta-glycerol phosphate but not by heparin. These properties of MAP kinase distinguish it from protein kinases previously described in the literature.

Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II.

Ribosomal protein S6 is a component of the eukaryotic 40S ribosomal subunit that becomes phosphorylated on multiple serine residues in response to a variety of mitogens, including insulin, growth factors, and transforming proteins of many oncogenic viruses. Recently, an activated S6 kinase (S6 K II) has been purified to homogeneity from Xenopus eggs, and characterized immunologically and at the molecular level. Purified S6 K II can be deactivated in vitro by incubation with either protein phosphatase 1 or protein phosphatase 2A. Reactivation and phosphorylation of S6 K II occurs in vitro with an insulin-stimulated microtubule-associated protein-2 (MAP-2) protein kinase which is itself a phosphoprotein that can be deactivated by protein phosphatase 2A. These studies suggest that a step in insulin signalling involves sequential activation by phosphorylation of at least two serine/threonine protein kinases.

Insulin-stimulated microtubule-associated protein kinase is phosphorylated on tyrosine and threonine in vivo.

Exposure of 3T3-L1 cells to insulin stimulates a soluble, serine(threonine)-specific protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) in vitro. The enzyme, termed MAP kinase, was isolated from insulin-treated or control cells radiolabeled with 32Pi. A 40-kDa phosphoprotein was found to elute in exact correspondence with enzymatic activity during hydrophobic interaction and gel filtration chromatography of extracts from cells stimulated with insulin. Both MAP kinase activity and the phosphoprotein were absent in fractions prepared from untreated cells. The 32P incorporated into the 40-kDa protein was stable during treatment with alkali. Phospho amino acid analysis confirmed that the radiolabel was primarily incorporated into phosphotyrosine and to a lesser extent phosphothreonine. In addition, MAP kinase was incompletely but specifically adsorbed by antibodies to phosphotyrosine. We conclude, based on these data and additional studies from this laboratory, that MAP kinase is phosphorylated on tyrosine in vivo. The data are consistent with the possibility that MAP kinase may be a substrate for the insulin receptor or another insulin-regulated tyrosine kinase.

Insulin-stimulated microtubule associated protein kinase is detectable by analytical gel chromatography as a 35-kDa protein in myocytes, adipocytes, and hepatocytes.

Insulin stimulates a novel Ser/Thr kinase, which phosphorylates microtubule associated protein-2 (MAP-2) in vitro. MAP kinase was studied in cell models of the principal insulin responsive tissues using analytical fast-protein liquid chromatography for partial purification of the enzyme. Stimulation of MAP kinase (1.3- to 2-fold) by insulin was readily detected in BC3H1 smooth and 23A2 skeletal muscle cells; 3T3-L1 adipocytes; and isolated rat hepatocytes and adipocytes. No phosphatase activity was detectable under the assay conditions used, proving that stimulation of a kinase, not inhibition of a phosphatase, is responsible for the increased incorporation of 32PO4 catalyzed by supernatants from insulin-treated 3T3-L1 cells. In H4 hepatoma cells, stimulation of MAP kinase was much less evident after gel filtration in comparison to the other cell types. The activated enzyme present in supernatants from insulin-treated cells migrated as a single peak of approximately 35 kDa apparent molecular mass (except in the case of isolated hepatocytes in which a shoulder was present). These results suggest that the insulin-stimulatable MAP kinase may be ubiquitous in insulin responsive cells.

Rapid stimulation by insulin of a serine/threonine kinase in 3T3-L1 adipocytes that phosphorylates microtubule-associated protein 2 in vitro.

Insulin treatment (Kact, 5 X 10(-9) M) of serum-starved 3T3-L1 adipocytes stimulates a soluble serine/threonine kinase that catalyzes phosphorylation of microtubule-associated protein 2 (MAP-2) in vitro. Maximal activation of MAP-2 kinase activity by 80 nM insulin was observed after 10 min of hormonal stimulation, prior to maximal stimulation of S6 kinase activity (20 min). The insulin-stimulatable MAP-2 kinase activity is not adsorbed to phosphocellulose, whereas the principal S6 kinase activity is retained and elutes at approximately 0.5 M NaCl. The insulin-stimulatable MAP-2 kinase is less stable during incubation at 30 degrees C than S6 kinase activity. Inclusion of phosphatase inhibitors decreases the rate at which the stimulated MAP-2 kinase activity is lost from extract supernatants incubated at 30 degrees C. p-Nitrophenyl phosphate is more effective than DL-phosphotyrosine, whereas DL-phosphoserine is without effect at the concentration used (40 mM). The difference in MAP-2 kinase activity in extract supernatants from control and insulin-treated cells is also preserved after rapid chromatography on Sephadex G-25. These results show that a soluble serine/threonine kinase is rapidly activated by insulin, possibly by phosphorylation of either the kinase itself or an interacting modulator.

An analysis of the influence of thyroid hormone on the synthesis of proteins in the tail fin of bullfrog tadpoles.

By incubation of explants of tail fin from tadpoles of Rana catesbeiana in a solution of 35S-methionine for 4 h, newly synthesized proteins were labeled isotopically. After separation by two-dimensional polyacrylamide gel electrophoresis, those proteins were visualized by fluorography. Exposure of explants to culture medium containing thyroxine (T4) (150 nM) increased the incorporation of 35S-methionine into several proteins with 48 h. Effects of T4 on the relative abundance of two of these newly synthesized proteins were detected after 8 h of hormonal treatment. Very similar patterns of newly synthesized proteins were observed when proteins from explants of tail fin removed from tadpoles at metamorphic climax and immediately incubated with 35S-methionine were compared with proteins produced in fin derived from premetamorphic animals. These results are interpreted to indicate that both treatment of explants with T4 and elevation of endogenous levels of thyroid hormones during spontaneous metamorphosis increased the relative rates of synthesis of several identical proteins. The potential involvement of those proteins in early phases of metamorphic action which eventually lead to cell death and resorption is discussed.

Observations on the interaction of prolactin and thyroxine in the tail of the bullfrog tadpole.

Explants of tail fin from tadpoles of Rana catesbeiana maintained in tissue culture underwent resorption when exposed to a medium containing 150 nM thyroxine (T4). Increases in the specific activity of hexosaminidase (Hex) and of acid phosphatase (AP) were associated with this response. Ovine prolactin inhibited resorption of explants in a dose-responsive manner; however, prolactin had no influence on the specific activity of either Hex or AP cultured explants. This result contrasted with the effect of prolactin in vivo, where it inhibited both the resorption of the tail and the increase in specific activity of hydrolytic enzymes induced by T4. The actions of both hormones, when applied in vivo, were more pronounced in the tail fin than in the muscular core of the tail at the stage of regression tested, indicating that the failure of prolactin to influence enzyme induction in vitro did not result from an incapacity of the fin to respond to prolactin in this fashion. It is suggested that the inhibition of T4-induced enzymic activity by prolactin which is observed in the intact animal may come about in response to some related but indirect systemic action of prolactin that is lacking when explants are cultured in vitro. It is concluded that inhibition of the induction of hydrolytic enzymes is not an essential factor in the inhibitory effect of prolactin on the resorption of the tail fin.

Investigations on the role of cAMP in regulating resorption of the tail fin from tadpoles of Rana catesbeiana.

Conflicting reports have appeared regarding the role of cAMP in regulating resorption of the tadpole tail during anuran metamorphosis. That cyclic nucleotide has been suggested as a mediator of the effects of both the thyroid hormones and prolactin. We tested the effects of cAMP and its derivatives dibutyryl-cAMP and 8-bromo-cAMP on explants of tail fin from tadpoles of Rana catesbeiana maintained in tissue culture. Unmodified cAMP (0.1, 2 mM) did not influence resorption. Dibutyryl-cAMP (0.1, 1 mM) and 8-bromo-cAMP (1 mM) inhibited resorption of explants induced by thyroxine (T4). The phosphodiesterase inhibitor isobutylmethylxanthine similarly inhibited regression of explants cultured with T4. None of these agents affected the increase in specific activity of hexosaminidase brought about by T4. Although the effects of cAMP in antagonizing tail resorption were similar to those of prolactin, we found no direct effect of prolactin on levels of cAMP in cultured tail fin. Thus, the effects of prolactin appear not to be mediated by increased levels of cAMP. We conclude, however, that the elevation of cellular levels of cAMP does inhibit the resorptive action of T4.

Muscle proteins related to microtubule associated protein-2 are substrates for an insulin-stimulatable kinase.

High molecular weight muscle protein(s), present as a "doublet" (approximately 320 and approximately 290 kDa apparent molecular weight) in partially purified preparations of inhibitor-2 from rabbit skeletal muscle, and homogeneous bovine brain microtubule associated protein-2 are both in vitro substrates for a soluble insulin-stimulatable serine/threonine kinase in 3T3-L1 adipocytes. The high molecular weight muscle substrate "doublet" was specifically immunoprecipitated by affinity-purified anti-microtubule associated protein-2 antibody.