Environmental disruption of the circadian clock leads to altered sleep and immune responses in mouse.

In mammals, one of the most salient outputs of the circadian (daily) clock is the timing of the sleep-wake cycle. Modern industrialized society has led to a fundamental breakdown in the relationship between our endogenous timekeeping systems and the solar day, disrupting normal circadian rhythms. We have argued that disrupted circadian rhythms could lead to changes in allostatic load, and the capacity of organisms to respond to other environmental challenges. In this set of studies, we apply a model of circadian disruption characterized in our lab in which mice are housed in a 20h long day, with 10h of light and 10h of darkness. We explored the effects of this environmental disruption on sleep patterns, to establish if this model results in marked sleep deprivation. Given the interaction between circadian, sleep, and immune systems, we further probed if our model of circadian disruption also alters the innate immune response to peripheral bacterial endotoxin challenge. Our results demonstrate that this model of circadian disruption does not lead to marked sleep deprivation, but instead affects the timing and quality of sleep. We also show that while circadian disruption does not lead to basal changes in the immune markers we explored, the immune response is affected, both in the brain and the periphery. Together, our findings further strengthen the important role of the circadian timing system in sleep regulation and immune responses, and provide evidence that disrupting the circadian clock increases vulnerability to further environmental stressors, including immunological challenges.

Brain-derived neurotrophic factor Val66Met polymorphism modulates the effects of circadian desynchronization on activity and sleep in male mice.

Introduction: Understanding how environmental interact challenges with genetic predispositions modulate health and wellbeing is an important area of biomedical research. Circadian rhythms play an important role in coordinating the multitude of cellular and tissue processes that organisms use to predict and adapt to regular changes in the environment, and robust circadian rhythms contribute to optimal physiological and behavioral responses to challenge. However, artificial lighting and modern round-the-clock lifestyles can disrupt the circadian system, leading to desynchronization of clocks throughout the brain and body. When coupled with genetic predispositions, circadian desynchronization may compound negative outcomes. Polymorphisms in the brain-derived neurotrophic (BDNF) gene contribute to variations in neurobehavioral responses in humans, including impacts on sleep, with the common Val66Met polymorphism linked to several negative outcomes. Methods: We explored how the Val66Met polymorphism modulates the response to environmental circadian desynchronization (ECD) in a mouse model. ECD was induced by housing adult male mice in a 20 h light-dark cycle (LD10:10; 10 h light, 10 h dark). Sleep and circadian activity were recorded in homozygous (Met) mice and their wild-type (Val) littermates in a standard 24 h LD cycle (LD12:12), then again after 20, 40, and 60 days of ECD. Results: We found ECD significantly affected the sleep/wake timing in Val mice, however, Met mice maintained appropriate sleep timing after 20 days ECD, but not after 40 and 60 days of ECD. In addition, the rise in delta power at lights on was absent in Val mice but was maintained in Met mice. To elucidate the circadian and homeostatic contribution to disrupted sleep, mice were sleep deprived by gentle handling in LD12:12 and after 20 days in ECD. Following 6 h of sleep deprivation delta power was increased for both Val and Met mice in LD12:12 and ECD conditions. However, the time constant was significantly longer in the Val mice during ECD compared to LD12:12, suggesting a functioning but altered sleep homeostat. Discussion: These data suggest the Val66Met mutation is associated with an ability to resist the effects of LD10:10, which may result in carriers suffering fewer negative impacts of ECD.

Estradiol influences adenosinergic signaling and nonrapid eye movement sleep need in adult female rats.

Gonadal steroids and gender are risk factors for sleep disruptions and insomnia in women. However, the relationship between ovarian steroids and sleep is poorly understood. In rodent models, estradiol (E2) suppresses sleep in females suggesting that E2 may reduce homeostatic sleep need. The current study investigates whether E2 decreases sleep need and the potential mechanisms that govern E2 suppression of sleep. Our previous findings suggest that the median preoptic nucleus (MnPO) is a key nexus for E2 action on sleep. Using behavioral, neurochemical, and pharmacological approaches, we tested whether (1) E2 influenced the sleep homeostat and (2) E2 influenced adenosine signaling in the MnPO of adult female rats. In both unrestricted baseline sleep and recovery sleep from 6-h sleep deprivation, E2 significantly reduced nonrapid eye movement (NREM) sleep-delta power, NREM-slow wave activity (NREM-SWA, 0.5-4.0 Hz), and NREM-delta energy suggesting that E2 decreases homeostatic sleep need. However, coordinated with E2-induced changes in physiological markers of homeostatic sleep was a marked increase in MnPO extracellular adenosine (a molecular marker of homeostatic sleep need) during unrestricted and recovery sleep in E2-treated but not oil control animals. While these results seemed contradictory, systemically administered E2 blocked the ability of CGS-21680 (adenosine A2A receptor agonist) microinjected into the MnPO to increase NREM sleep suggesting that E2 may block adenosine signaling. Together, these findings provide evidence that E2 may attenuate the local effects of the A2A receptors in the MnPO, which in turn may underlie estrogenic suppression of sleep behavior as well as changes in homeostatic sleep need.

Role of corticosterone in altered neurobehavioral responses to acute stress in a model of compromised hypothalamic-pituitary-adrenal axis function.

An organism's capacity to cope with stressful experiences is dependent on its ability to appropriately engage central and peripheral systems, such as the hypothalamic-pituitary-adrenal (HPA) axis, to adapt to changing environmental demands. The HPA axis is a primary neuroendocrine mediator of neural and behavioral responses to stress, and dysfunction of this system is linked to increased risk for developing mental health disorders such as depression, anxiety, and post-traumatic stress disorder. However, the mechanisms by which dysregulated HPA function results in abnormal behavioral responses to stress are poorly understood. Here, we tested how corticosterone (CORT)-induced HPA axis disruption affects behavioral responses to stress in male C57BL/6 N mice, and probed correlates of these behaviors in the brain. We show that chronic HPA disruption blunts acute stress-induced grooming and rearing behaviors in the open field test, effects which were accompanied by decreased FOS immunoreactivity in the paraventricular nucleus of the hypothalamus (PVH) and paraventricular nucleus of the thalamus (PVT). Blockade of CORT secretion with metyrapone injection prior to acute stress did not recapitulate the effects of chronic HPA disruption on open field behavior, and acute CORT replacement did not rescue normal behavioral stress responses following chronic HPA disruption. This suggests that under acute conditions, CORT is not necessary for these responses normally, nor sufficient to rescue the deficits of chronic HPA dysregulation. Together, these findings support the hypothesis that chronic HPA dysregulation causes adaptation in stress-related brain circuits and demonstrate that these changes can influence an organism's behavioral response to stress exposure.

Characterization of flexible ECoG electrode arrays for chronic recording in awake rats.

We developed a 64-channel flexible polyimide ECoG electrode array and characterized its performance for long-term implantation, chronic cortical recording and high resolution mapping of surface-evoked potentials in awake rats. To achieve the longest possible recording periods, the flexibility of the electrode array, adhesion between the metals and carrier substrate, and biocompatibility were critical for maintaining the signal integrity. Experimental testing of thin film adhesion was applied to a gold-polyimide system in order to characterize relative interfacial fracture energies for several different adhesion layers, yielding an increase in overall device reliability. We tested several different adhesion techniques including the following: gold alone without an adhesion layer, titanium-tungsten, tantalum and chromium. We found titanium-tungsten to be a suitable adhesion layer considering the biocompatibility requirements as well as stability and delamination resistance. While chromium and tantalum produced stronger gold adhesion, concerns over biocompatibility of these materials require further testing. We implanted the polyimide ECoG electrode arrays through a slit made in the skull of rats and recorded cortical surface evoked responses. The arrays performed reliably over a period of at least 100 days and signals compared well with traditional screw electrodes, with better high frequency response characteristics. Since the ultimate goal of chronically implanted electrode arrays is for neural prosthetic devices that need to last many decades, other adhesion layers that would prove safe for implantation may be tested in the same way in order to improve the device reliability.

Vascular compliance limits during sleep deprivation and recovery sleep.

STUDY OBJECTIVES: Our previous studies showed that evoked hemodynamic responses are smaller during wake compared to sleep; suggesting neural activity is associated with vascular expansion and decreased compliance. We explored whether prolonged activity during sleep deprivation may exacerbate vascular expansion and blunt hemodynamic responses. DESIGN: Evoked auditory responses were generated with periodic 65 dB speaker clicks over a 72-h period and measured with cortical electrodes. Evoked hemodynamic responses were measured simultaneously with optical techniques using three light-emitting diodes, and a photodiode. SETTING: Animals were housed in separate 30×30×80 cm enclosures, tethered to a commutator system and maintained on a 12-h light/dark cycle. Food and water were available ad libitum. PATIENTS OR PARTICIPANTS: Seven adult female Sprague-Dawley rats. INTERVENTIONS: Following a 24-h baseline recording, sleep deprivation was initiated for 0 to 10 h by gentle handling, followed by a 24-h recovery sleep recording. Evoked electrical and hemodynamic responses were measured before, during, and after sleep deprivation. MEASUREMENTS AND RESULTS: Following deprivation, evoked hemodynamic amplitudes were blunted. Steady-state oxyhemoglobin concentration increased during deprivation and remained high during the initial recovery period before returning to baseline levels after approximately 9-h. CONCLUSIONS: Sleep deprivation resulted in blood vessel expansion and decreased compliance while lower basal neural activity during recovery sleep may allow blood vessel compliance to recover. Chronic sleep restriction or sleep deprivation could push the vasculature to critical levels, limiting blood delivery, and leading to metabolic deficits with the potential for neural trauma.

Cortical evoked responses associated with arousal from sleep.

STUDY OBJECTIVE: To determine if low-level intermittent auditory stimuli have the potential to disrupt sleep during 24-h recordings, we assessed arousal occurrence to varying stimulus intensities. Additionally, if stimulus-generated evoked response potential (ERP) components provide a metric of underlying cortical state, then a particular ERP structure may precede an arousal. DESIGN: Physiological electrodes measuring EEG, EKG, and EMG were implanted into 5 adult female Sprague-Dawley rats. We delivered auditory stimuli of varying intensities (50-75 dBa sound pressure level SPL) at random intervals of 6-12 s over a 24-hour period. Recordings were divided into 2-s epochs and scored for sleep/wake state. Following each stimulus, we identified whether the animal stayed asleep or woke. We then sorted the stimuli depending on prior and post-stimulus state, and measured ERP components. RESULTS: Auditory stimuli did not produce a significant increase in the number of arousals compared to silent control periods. Overall, arousal from REM sleep occurred more often compared to quiet sleep. ERPs preceding an arousal had decreased mean area and shorter N1 latency. CONCLUSION: Low level auditory stimuli did not fragment animal sleep since we observed no significant change in arousal occurrence. Arousals that occurred within 4 s of a stimulus exhibited an ERP mean area and latency had features similar to ERPs generated during wake, indicating that the underlying cortical tissue state may contribute to physiological conditions required for arousal.

State-dependent changes in cortical gain control as measured by auditory evoked responses to varying intensity stimuli.

STUDY OBJECTIVES: Auditory evoked potential (AEP) components correspond to sequential activation of brain structures within the auditory pathway and reveal neural activity during sensory processing. To investigate state-dependent modulation of stimulus intensity response profiles within different brain structures, we assessed AEP components across both stimulus intensity and state. DESIGN: We implanted adult female Sprague-Dawley rats (N = 6) with electrodes to measure EEG, EKG, and EMG. Intermittent auditory stimuli (6-12 s) varying from 50 to 75 dBa were delivered over a 24-h period. Data were parsed into 2-s epochs and scored for wake/sleep state. RESULTS: All AEP components increased in amplitude with increased stimulus intensity during wake. During quiet sleep, however, only the early latency response (ELR) showed this relationship, while the middle latency response (MLR) increased at the highest 75 dBa intensity, and the late latency response (LLR) showed no significant change across the stimulus intensities tested. During rapid eye movement sleep (REM), both ELR and LLR increased, similar to wake, but MLR was severely attenuated. CONCLUSIONS: Stimulation intensity and the corresponding AEP response profile were dependent on both brain structure and sleep state. Lower brain structures maintained stimulus intensity and neural response relationships during sleep. This relationship was not observed in the cortex, implying state-dependent modification of stimulus intensity coding. Since AEP amplitude is not modulated by stimulus intensity during sleep, differences between paired 75/50 dBa stimuli could be used to determine state better than individual intensities.