Dopamine and Wakefulness: Pharmacology, Genetics, and Circuitry.

Over the period of decades in the mid to late twentieth century, arousal-promoting functions were attributed to neuromodulators including serotonin, hypocretin, histamine, and noradrenaline. For some time, a relatively minor role in regulating sleep and wake states was ascribed to dopamine and the dopamine-producing cells of the ventral tegmental area, despite the fact that dopaminergic signaling is a major target, if not the primary target, for wake-promoting agents. In recent years, due to observations from human genetic studies, pharmacogenetic studies in animal models, and the increasingly sophisticated methods used to manipulate the nervous systems of experimental animals, it has become clear that dopaminergic signaling is central to the regulation of arousal. This chapter reviews this central role of dopaminergic signaling, and in particular its antagonistic interaction with adenosinergic signaling, in maintaining vigilance and in the response to wake-promoting therapeutics.

Beta EEG reflects sensory processing in active wakefulness and homeostatic sleep drive in quiet wakefulness.

Markers of sleep drive (<10 Hz; slow-wave activity and theta) have been identified in the course of slow-wave sleep and wakefulness. So far, higher frequencies in the waking electroencephalogram have not been examined thoroughly as a function of sleep drive. Here, electroencephalogram dynamics were measured in epochs of active wake (wake characterized by high muscle tone) or quiet wake (wake characterized by low muscle tone). It was hypothesized that the higher beta oscillations (15-35 Hz, measured by local field potential and electroencephalography) represent fundamentally different processes in active wake and quiet wake. In active wake, sensory stimulation elevated beta activity in parallel with gamma (80-90 Hz) activity, indicative of cognitive processing. In quiet wake, beta activity paralleled slow-wave activity (1-4 Hz) and theta (5-8 Hz) in tracking sleep need. Cerebral lactate concentration, a measure of cerebral glucose utilization, increased during active wake whereas it declined during quiet wake. Mathematical modelling of state-dependent dynamics of cortical lactate concentration was more precisely predictive when quiet wake and active wake were included as two distinct substates rather than a uniform state of wakefulness. The extent to which lactate concentration declined in quiet wake and increased in active wake was proportionate to the amount of beta activity. These data distinguish quiet wake from active wake. Quiet wake, particularly when characterized by beta activity, is permissive to metabolic and electrophysiological changes that occur in slow-wave sleep. These data urge further studies on state-dependent beta oscillations across species.

GABAB agonism promotes sleep and reduces cataplexy in murine narcolepsy.

γ-Hydroxybutyrate (GHB) is an approved therapeutic for the excessive sleepiness and sudden loss of muscle tone (cataplexy) characteristic of narcolepsy. The mechanism of action for these therapeutic effects is hypothesized to be GABAB receptor dependent. We evaluated the effects of chronic administration of GHB and the GABAB agonist R-baclofen (R-BAC) on arousal state and cataplexy in two models of narcolepsy: orexin/ataxin-3 (Atax) and orexin/tTA; TetO diphtheria toxin mice (DTA). Mice were implanted for EEG/EMG monitoring and dosed with GHB (150 mg/kg), R-BAC (2.8 mg/kg), or vehicle (VEH) bid for 15 d-a treatment paradigm designed to model the twice nightly GHB dosing regimen used by human narcoleptics. In both models, R-BAC increased NREM sleep time, intensity, and consolidation during the light period; wake bout duration increased and cataplexy decreased during the subsequent dark period. GHB did not increase NREM sleep consolidation or duration, although NREM delta power increased in the first hour after dosing. Cataplexy decreased from baseline in 57 and 86% of mice after GHB and R-BAC, respectively, whereas cataplexy increased in 79% of the mice after VEH. At the doses tested, R-BAC suppressed cataplexy to a greater extent than GHB. These results suggest utility of R-BAC-based therapeutics for narcolepsy.

TNFα G308A polymorphism is associated with resilience to sleep deprivation-induced psychomotor vigilance performance impairment in healthy young adults.

Cytokines such as TNFα play an integral role in sleep/wake regulation and have recently been hypothesized to be involved in cognitive impairment due to sleep deprivation. We examined the effect of a guanine to adenine substitution at position 308 in the TNFα gene (TNFα G308A) on psychomotor vigilance performance impairment during total sleep deprivation. A total of 88 healthy women and men (ages 22-40) participated in one of five laboratory total sleep deprivation experiments. Performance on a psychomotor vigilance test (PVT) was measured every 2-3h. The TNFα 308A allele, which is less common than the 308G allele, was associated with greater resilience to psychomotor vigilance performance impairment during total sleep deprivation (regardless of time of day), and also provided a small performance benefit at baseline. The effect of genotype on resilience persisted when controlling for between-subjects differences in age, gender, race/ethnicity, and baseline sleep duration. The TNFα G308A polymorphism predicted less than 10% of the overall between-subjects variance in performance impairment during sleep deprivation. Nonetheless, the differential effect of the polymorphism at the peak of performance impairment was more than 50% of median performance impairment at that time, which is sizeable compared to the effects of other genotypes reported in the literature. Our findings provided evidence for a role of TNFα in the effects of sleep deprivation on psychomotor vigilance performance. Furthermore, the TNFα G308A polymorphism may have predictive potential in a biomarker panel for the assessment of resilience to psychomotor vigilance performance impairment due to sleep deprivation.

Sleep slow-wave activity regulates cerebral glycolytic metabolism.

Non-rapid eye movement sleep (NREMS) onset is characterized by a reduction in cerebral metabolism and an increase in slow waves, 1-4-Hz oscillations between relatively depolarized and hyperpolarized states in the cerebral cortex. The metabolic consequences of slow-wave activity (SWA) at the cellular level remain uncertain. We sought to determine whether SWA modulates the rate of glycolysis within the cerebral cortex. The real-time measurement of lactate concentration in the mouse cerebral cortex demonstrates that it increases during enforced wakefulness. In spontaneous sleep/wake cycles, lactate concentration builds during wakefulness and rapid eye movement sleep and declines during NREMS. The rate at which lactate concentration declines during NREMS is proportional to the magnitude of electroencephalographic (EEG) activity at frequencies of <10 Hz. The induction of 1-Hz oscillations, but not 10-Hz oscillations, in the electroencephalogram by optogenetic stimulation of cortical pyramidal cells during wakefulness triggers a decline in lactate concentration. We conclude that cerebral SWA promotes a decline in the rate of glycolysis in the cerebral cortex. These results demonstrate a cellular energetic function for sleep SWA, which may contribute to its restorative effects on brain function.

Chronic dietary supplementation with nicotinamide riboside reduces sleep need in the laboratory mouse.

Non-rapid eye movement sleep (NREMS) is accompanied by a reduction in cerebral glucose utilization. Enabling this metabolic change may be a central function of sleep. Since the reduction in glucose metabolism is inevitably accompanied by deceleration of downstream oxidation/reduction reactions involving nicotinamide adenine dinucleotide (NAD), we hypothesized a role for NAD in regulating the homeostatic dynamics of sleep at the biochemical level. We applied dietary nicotinamide riboside (NR), a NAD precursor, in a protocol known to improve neurological outcome measures in mice. Long-term (6-10 weeks) dietary supplementation with NR reduced the time that mice spent in NREMS by 17 percent and accelerated the rate of discharge of sleep need according to a mathematical model of sleep homeostasis (Process S). These findings suggest that increasing redox capacity by increasing nicotinamide availability reduces sleep need and increases the cortical capacity for energetically demanding high-frequency oscillations. In turn, this work demonstrates the impact of redox substrates on cortical circuit properties related to fatigue and sleep drive, implicating redox reactions in the homeostatic dynamics of cortical network events across sleep-wake cycles.

Effect of N-Acetylcysteine on Sleep: Impacts of Sex and Time of Day.

Non-rapid eye movement sleep (NREMS) is accompanied by a decrease in cerebral metabolism, which reduces the consumption of glucose as a fuel source and decreases the overall accumulation of oxidative stress in neural and peripheral tissues. Enabling this metabolic shift towards a reductive redox environment may be a central function of sleep. Therefore, biochemical manipulations that potentiate cellular antioxidant pathways may facilitate this function of sleep. N-acetylcysteine increases cellular antioxidant capacity by serving as a precursor to glutathione. In mice, we observed that intraperitoneal administration of N-acetylcysteine at a time of day when sleep drive is naturally high accelerated the onset of sleep and reduced NREMS delta power. Additionally, N-acetylcysteine administration suppressed slow and beta electroencephalographic (EEG) activities during quiet wake, further demonstrating the fatigue-inducing properties of antioxidants and the impact of redox balance on cortical circuit properties related to sleep drive. These results implicate redox reactions in the homeostatic dynamics of cortical network events across sleep/wake cycles, illustrating the value of timing antioxidant administration relative to sleep/wake cycles. A systematic review of the relevant literature, summarized herein, indicates that this "chronotherapeutic hypothesis" is unaddressed within the clinical literature on antioxidant therapy for brain disorders such as schizophrenia. We, therefore, advocate for studies that systematically address the relationship between the time of day at which an antioxidant therapy is administered relative to sleep/wake cycles and the therapeutic benefit of that antioxidant treatment in brain disorders.

A metabolic-transcriptional network links sleep and cellular energetics in the brain.

This review proposes a mechanistic link between cellular metabolic status, transcriptional regulatory changes and sleep. Sleep loss is associated with changes in cellular metabolic status in the brain. Metabolic sensors responsive to cellular metabolic status regulate the circadian clock transcriptional network. Modifications of the transcriptional activity of circadian clock genes affect sleep/wake state changes. Changes in sleep state reverse sleep loss-induced changes in cellular metabolic status. It is thus proposed that the regulation of circadian clock genes by cellular metabolic sensors is a critical intermediate step in the link between cellular metabolic status and sleep. Studies of this regulatory relationship may offer insights into the function of sleep at the cellular level.

Hypocretin/orexin and nociceptin/orphanin FQ coordinately regulate analgesia in a mouse model of stress-induced analgesia.

Stress-induced analgesia (SIA) is a key component of the defensive behavioral "fight-or-flight" response. Although the neural substrates of SIA are incompletely understood, previous studies have implicated the hypocretin/orexin (Hcrt) and nociceptin/orphanin FQ (N/OFQ) peptidergic systems in the regulation of SIA. Using immunohistochemistry in brain tissue from wild-type mice, we identified N/OFQ-containing fibers forming synaptic contacts with Hcrt neurons at both the light and electron microscopic levels. Patch clamp recordings in GFP-tagged mouse Hcrt neurons revealed that N/OFQ hyperpolarized, decreased input resistance, and blocked the firing of action potentials in Hcrt neurons. N/OFQ postsynaptic effects were consistent with opening of a G protein-regulated inwardly rectifying K+ (GIRK) channel. N/OFQ also modulated presynaptic release of GABA and glutamate onto Hcrt neurons in mouse hypothalamic slices. Orexin/ataxin-3 mice, in which the Hcrt neurons degenerate, did not exhibit SIA, although analgesia was induced by i.c.v. administration of Hcrt-1. N/OFQ blocked SIA in wild-type mice, while coadministration of Hcrt-1 overcame N/OFQ inhibition of SIA. These results establish what is, to our knowledge, a novel interaction between the N/OFQ and Hcrt systems in which the corticotropin-releasing factor and N/OFQ systems coordinately modulate the Hcrt neurons to regulate SIA.

Cerebral microglia mediate sleep/wake and neuroinflammatory effects of methamphetamine.

Methamphetamine and modafinil exert their wake-promoting effects by elevating monoaminergic tone. The severity of hypersomnolence that occurs subsequent to induced wakefulness differs between these two agents. Microglia detects and modulates CNS reactions to agents such as D-methamphetamine that induce cellular stress. We therefore hypothesized that changes in the sleep/wake cycle that occur subsequent to administration of D-methamphetamine are modulated by cerebral microglia. In CD11b-herpes thymidine kinase transgenic mice (CD11b-TK(mt-30)), activation of the inducible transgene by intracerebroventricular (icv) ganciclovir results in toxicity to CD11b-positive cells (i.e. microglia), thereby reducing cerebral microglial cell counts. CD11b-TK(mt-30)and wild type mice were subjected to chronic icv ganciclovir or vehicle administration with subcutaneous mini-osmotic pumps. D-methamphetamine (1 and 2 mg/kg), modafinil (30 and 100 mg/kg) and vehicle were administered intraperitoneally to these animals. In CD11b-TK(mt-30) mice, but not wild type, icv infusion of ganciclovir reduced the duration of wake produced by D-methamphetamine at 2 mg/kg by nearly 1h. Nitric oxide synthase (NOS) activity, studied ex vivo, and NOS expression were elevated in CD11b-positive cerebral microglia from wild type mice acutely exposed to d-methamphetamine. Additionally, CD11b-positive microglia, but not other cerebral cell populations, exhibited changes in sleep-regulatory cytokine expression in response to d-METH. Finally, CD11b-positive microglia exposed to d-methamphetamine in vitro exhibited increased NOS activity relative to pharmacologically-naïve cells. CD11b-positive microglia from the brains of neuronal NOS (nNOS)-knockout mice failed to exhibit this effect. We propose that the effects of D-METH on sleep/wake cycles are mediated in part by actions on microglia, including possibly nNOS activity and cytokine synthesis.

Identification of a population of sleep-active cerebral cortex neurons.

The presence of large-amplitude, slow waves in the EEG is a primary characteristic that distinguishes cerebral activity during sleep from that which occurs during wakefulness. Although sleep-active neurons have been identified in other brain areas, neurons that are specifically activated during slow-wave sleep have not previously been described in the cerebral cortex. We have identified a population of cells in the cortex that is activated during sleep in three mammalian species. These cortical neurons are a subset of GABAergic interneurons that express neuronal NOS (nNOS). Because Fos expression in these sleep-active, nNOS-immunoreactive (nNOS-ir) neurons parallels changes in the intensity of slow-wave activity in the EEG, and these neurons are innvervated by neurotransmitter systems previously implicated in sleep/wake control, cortical nNOS-ir neurons may be part of the neurobiological substrate that underlies homeostatic sleep regulation.

Multi-Modal Regulation of Circadian Physiology by Interactive Features of Biological Clocks.

The circadian clock is a fundamental biological timing mechanism that generates nearly 24 h rhythms of physiology and behaviors, including sleep/wake cycles, hormone secretion, and metabolism. Evolutionarily, the endogenous clock is thought to confer living organisms, including humans, with survival benefits by adapting internal rhythms to the day and night cycles of the local environment. Mirroring the evolutionary fitness bestowed by the circadian clock, daily mismatches between the internal body clock and environmental cycles, such as irregular work (e.g., night shift work) and life schedules (e.g., jet lag, mistimed eating), have been recognized to increase the risk of cardiac, metabolic, and neurological diseases. Moreover, increasing numbers of studies with cellular and animal models have detected the presence of functional circadian oscillators at multiple levels, ranging from individual neurons and fibroblasts to brain and peripheral organs. These oscillators are tightly coupled to timely modulate cellular and bodily responses to physiological and metabolic cues. In this review, we will discuss the roles of central and peripheral clocks in physiology and diseases, highlighting the dynamic regulatory interactions between circadian timing systems and multiple metabolic factors.

Effect of Dietary Nitrate Supplementation on Sleep in Chronic Obstructive Pulmonary Disease Patients.

PURPOSE: Poor sleep quality in chronic obstructive pulmonary disease (COPD) is a result of oxygen desaturation secondary to compromised lung function. Nitrate supplementation with dietary beetroot juice is known to elevate plasma nitrate and to increase the efficiency of oxygen utilization in non-COPD individuals; whether it is of therapeutic benefit for sleep quality in COPD has not been reported. PATIENTS AND METHODS: In a counterbalanced within-subjects design involving 15 COPD patients as subjects, the subjects consumed either beetroot juice containing nitrate (BJ; ∼6.2 mmol NO3 -) or placebo (NO3 - -depleted juice) immediately before a night of polysomnographic monitoring. Nitrate was measured in plasma collected immediately after waking. RESULTS: While BJ consumption had no effect on the amount of time spent in any sleep stages, wake-to-N2 transitions and direct wake-to-rapid eye movement sleep (REMS) transitions, hallmarks of disordered sleep, were less frequent on the BJ night than on the placebo night. In the last two hours of the BJ night, percent time in REMS increased and delta power during deep (N3) non-REMS decreased, relative to the placebo night. Collectively, the reduced frequency of atypical transitions and the normalization of non-REMS/REMS dynamics after BJ are indicative of an improvement of sleep quality. BJ also resulted in sustained elevation of peripheral oxygen saturation (SpO2), during episodes of wake after sleep onset. Plasma nitrate was elevated nearly tenfold on the morning after BJ relative to placebo. CONCLUSION: BJ has a normalizing effect on disordered sleep in COPD, which may be related to improved oxygen delivery. CLINICAL TRIAL REGISTRATION: The activities of the Regional Committees for Medical and Health Research Ethics (REC) are founded on the Norwegian law on research ethics and medical research. This study was approved by NTNU/REK midt, Det medisinske fakultet, Postboks 8905, 7491 Trondheim (REK midt 2016/1360).

Diurnal changes in perineuronal nets and parvalbumin neurons in the rat medial prefrontal cortex.

Perineuronal nets (PNNs) surrounding fast-spiking, parvalbumin (PV) interneurons provide excitatory:inhibitory balance, which is impaired in several disorders associated with altered diurnal rhythms, yet few studies have examined diurnal rhythms of PNNs or PV cells. We measured the intensity and number of PV cells and PNNs labeled with Wisteria floribunda agglutinin (WFA) and also the oxidative stress marker 8-oxo-deoxyguanosine (8-oxo-dG) in rat prelimbic medial prefrontal cortex (mPFC) at Zeitgeber times (ZT) ZT0 (lights-on, inactive phase), ZT6 (mid-inactive phase), ZT12 (lights-off, active phase), and ZT18 (mid-active phase). Relative to ZT0, the intensities of PNN and PV labeling were increased in the dark (active) phase compared with the light (inactive) phase. The intensity of 8-oxo-dG was decreased from ZT0 at all times (ZT6,12,18). We also measured GAD 65/67 and vGLUT1 puncta apposed to PV cells with and without PNNs. There were more excitatory puncta on PV cells with PNNs at ZT18 vs. ZT6, but no changes in PV cells without PNNs and no changes in inhibitory puncta. Whole-cell slice recordings in fast-spiking (PV) cells with PNNs showed an increased ratio of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor:N-methyl-D-aspartate receptor (AMPA: NMDA) at ZT18 vs. ZT6. The number of PV cells and PV/PNN cells containing orthodenticle homeobox 2 (OTX2), which maintains PNNs, showed a strong trend toward an increase from ZT6 to ZT18. Diurnal fluctuations in PNNs and PV cells are expected to alter cortical excitatory:inhibitory balance and provide new insights into treatments for diseases impacted by disturbances in sleep and circadian rhythms.

Thyrotropin-releasing hormone increases behavioral arousal through modulation of hypocretin/orexin neurons.

Thyrotropin-releasing hormone (TRH) has previously been shown to promote wakefulness and to induce arousal from hibernation. Expression of TRH-R1 (TRH receptor 1) is enriched in the tuberal and lateral hypothalamic area (LHA), brain regions in which the hypocretin/orexin (Hcrt) cells are located. Because the Hcrt system is implicated in sleep/wake control, we hypothesized that TRH provides modulatory input to the Hcrt cells. In vitro electrophysiological studies showed that bath application of TRH caused concentration-dependent membrane depolarization, decreased input resistance, and increased firing rate of identified Hcrt neurons. In the presence of tetrodotoxin, TRH induced inward currents that were associated with a decrease in frequency, but not amplitude, of miniature postsynaptic currents (PSCs). Ion substitution experiments suggested that the TRH-induced inward current was mediated in part by Ca(2+) influx. Although TRH did not significantly alter either the frequency or amplitude of spontaneous excitatory PSCs, TRH (100 nm) increased the frequency of spontaneous inhibitory PSCs by twofold without affecting the amplitude of these events, indicating increased presynaptic GABA release onto Hcrt neurons. In contrast, TRH significantly reduced the frequency, but not amplitude, of miniature excitatory PSCs without affecting miniature inhibitory PSC frequency or amplitude, indicating that TRH also reduces the probability of glutamate release onto Hcrt neurons. When injected into the LHA, TRH increased locomotor activity in wild-type mice but not in orexin/ataxin-3 mice in which the Hcrt neurons degenerate postnatally. Together, these results are consistent with the hypothesis that TRH modulates behavioral arousal, in part, through the Hcrt system.

A Role for Astroglial Calcium in Mammalian Sleep and Sleep Regulation.

Mammalian sleep expression and regulation have historically been thought to reflect the activity of neurons. Changes in other brain cells (glia) across the sleep-wake cycle and their role in sleep regulation are comparatively unexplored. We show that sleep and wakefulness are accompanied by state-dependent changes in astroglial activity. Using a miniature microscope in freely behaving mice and a two-photon microscope in head-fixed, unanesthetized mice, we show that astroglial calcium signals are highest in wake and lowest in sleep and are most pronounced in astroglial processes. We also find that astroglial calcium signals during non-rapid eye movement sleep change in proportion to sleep need. In contrast to neurons, astrocytes become less synchronized during non-rapid eye movement sleep after sleep deprivation at the network and single-cell level. Finally, we show that conditionally reducing intracellular calcium in astrocytes impairs the homeostatic response to sleep deprivation. Thus, astroglial calcium activity changes dynamically across vigilance states, is proportional to sleep need, and is a component of the sleep homeostat.

DRD2 C957T genotype modulates the time-on-task effect during total sleep deprivation.

Total sleep deprivation (TSD) and time-on-task (TOT), especially in combination, increase cognitive instability and cause performance impairment. There are large inter-individual differences in TSD and TOT effects which, in part, have a genetic basis. Here, we show that the dopamine receptor D2 C957T genetic polymorphism predicts the magnitude of the TOT effect on a psychomotor vigilance test (PVT) during 38 h of TSD. This finding indicates that dopamine availability in the striatum, where the dopamine receptor D2 is most prevalent, influences the TOT effect, suggesting a role for dopaminergic pathways in sustained attention deficits during sleep loss.

Cognitive function and brain plasticity in a rat model of shift work: role of daily rhythms, sleep and glucocorticoids.

Many occupations require operations during the night-time when the internal circadian clock promotes sleep, in many cases resulting in impairments in cognitive performance and brain functioning. Here, we use a rat model to attempt to identify the biological mechanisms underlying such impaired performance. Rats were exposed to forced activity, either in their rest-phase (simulating night-shift work; rest work) or in their active-phase (simulating day-shift work; active work). Sleep, wakefulness and body temperature rhythm were monitored throughout. Following three work shifts, spatial memory performance was tested on the Morris Water Maze task. After 4 weeks washout, the work protocol was repeated, and blood and brain tissue collected. Simulated night-shift work impaired spatial memory and altered biochemical markers of cerebral cortical protein synthesis. Measures of daily rhythm strength were blunted, and sleep drive increased. Individual variation in the data suggested differences in shift work tolerance. Hierarchical regression analyses revealed that type of work, changes in daily rhythmicity and changes in sleep drive predict spatial memory performance and expression of brain protein synthesis regulators. Moreover, serum corticosterone levels predicted expression of brain protein synthesis regulators. These findings open new research avenues into the biological mechanisms that underlie individual variation in shift work tolerance.

Sleep deprivation effects on circadian clock gene expression in the cerebral cortex parallel electroencephalographic differences among mouse strains.

Sleep deprivation (SD) results in increased electroencephalographic (EEG) delta power during subsequent non-rapid eye movement sleep (NREMS) and is associated with changes in the expression of circadian clock-related genes in the cerebral cortex. The increase of NREMS delta power as a function of previous wake duration varies among inbred mouse strains. We sought to determine whether SD-dependent changes in circadian clock gene expression parallel this strain difference described previously at the EEG level. The effects of enforced wakefulness of incremental durations of up to 6 h on the expression of circadian clock genes (bmal1, clock, cry1, cry2, csnk1epsilon, npas2, per1, and per2) were assessed in AKR/J, C57BL/6J, and DBA/2J mice, three strains that exhibit distinct EEG responses to SD. Cortical expression of clock genes subsequent to SD was proportional to the increase in delta power that occurs in inbred strains: the strain that exhibits the most robust EEG response to SD (AKR/J) exhibited dramatic increases in expression of bmal1, clock, cry2, csnkIepsilon, and npas2, whereas the strain with the least robust response to SD (DBA/2) exhibited either no change or a decrease in expression of these genes and cry1. The effect of SD on circadian clock gene expression was maintained in mice in which both of the cryptochrome genes were genetically inactivated. cry1 and cry2 appear to be redundant in sleep regulation as elimination of either of these genes did not result in a significant deficit in sleep homeostasis. These data demonstrate transcriptional regulatory correlates to previously described strain differences at the EEG level and raise the possibility that genetic differences underlying circadian clock gene expression may drive the EEG differences among these strains.

Sleep disruption elevates oxidative stress in parvalbumin-positive cells of the rat cerebral cortex.

We used a novel automated sleep disruption (SD) apparatus to determine the impact of SD on sleep and molecular markers of oxidative stress in parvalbumin (PV) neurons in the rat prefrontal cortex (PFC). Rats were subjected to two 6 hr SD sessions from zeitgeber time (ZT) 0 to ZT6, one by the gentle handling method and the other by an automated agitator running the length of the rat's home cage floor (a novel SD method). The same rats were later subjected to a 12 hr SD session from ZT0 to ZT12. Sleep was disrupted with both methods, although rats slept less during gentle handling than during the automated condition. Immediately after both SD sessions, rats displayed compensatory sleep characterized by elevated slow-wave activity. We measured in the prelimbic prefrontal cortex (prelimbic PFC; 6 and 12 hr SD) and orbital frontal cortex (12 hr SD) the intensity of the oxidative stress marker, 8-oxo-2'-deoxyguanosine (8-oxo-dG) as well as the staining intensity of PV and the PV cell-associated perineuronal net marker, Wisteria floribunda agglutinin (WFA). In the prelimbic PFC, 6 hr SD increased the intensity of 8-oxo-dG, PV, and WFA. After 12 hr SD, the intensity of 8-oxo-dG was elevated in all neurons. PV intensity was elevated only in neurons colabeled with 8-oxo-dG or WFA, and no changes were found in WFA intensity. We conclude that in association with SD-induced sleep drive, PV neurons in the prelimbic PFC exhibit oxidative stress.