STUDY OBJECTIVES:Poor sleep is linked to adverse health outcomes. Animal studies suggest the gut microbiome may influence sleep, but human findings remain inconsistent. We examined associations of self-reported insomnia symptoms, daytime sleepiness, and actigraphy-measured sleep with gut microbiome diversity and composition in older adults. METHODS:We studied 869 Baltimore Longitudinal Study of Aging participants with self-reported sleep and shotgun metagenomic sequencing; 332 also had actigraphy. We tested associations of sleep with alpha diversity, beta diversity, and species composition using regression, PERMANOVA, and ANCOM-BC2, adjusting for age, sex, BMI, physical activity, education, and depressive symptoms. RESULTS:Participants had mean age 70.7 years; 54.8% were female and 66.9% White. Trouble falling asleep ≥5 times/week was associated with higher Shannon diversity (β=0.41 SD; 95% CI: 0.09, 0.73) and Pielou's evenness, but not richness metrics. No actigraphy-measured sleep variables were associated with alpha or beta diversity. Beta diversity analyses suggested excessive sleepiness (1-2 o 3-4 times/week) was associated with different microbial composition, though variance explained was small. In species-level analyses, frequent insomnia symptoms or excessive sleepiness were associated with depleted or undetected Eubacterium sp. CAG:251. In exploratory actigraphy models, each doubling of sleep efficiency was associated with higher Eubacterium sp. CAG:251 prevalence (PR=2.15; 95% CI: 1.47, 3.14), while each 30-minute increase in wake after sleep onset was associated with lower prevalence (PR=0.49; 95% CI: 0.29, 0.81). CONCLUSIONS:Global diversity findings were limited and inconsistent, whereas subjective and objective sleep disturbances converged on Eubacterium sp. CAG:251. Findings are exploratory and require longitudinal replication.

BACKGROUND:Sleep disturbances and cardiovascular disease are common and often co-occur in middle-aged and older adults, but less is known about associations of sleep with cardiorespiratory fitness (CRF) and energy efficiency in these populations. We examined cross-sectional associations of accelerometer-derived sleep metrics with CRF, walking energetics, and resting metabolic rate (RMR), and explored whether associations were moderated by age, sex, and race. METHODS:We studied 263 participants from the Baltimore Longitudinal Study of Aging (mean age 72.7 ± 10.1 years, 53.6% women). Predictors included total sleep time (TST), sleep efficiency (SE), sleep onset latency, wake after sleep onset, and average wake bout length (WBL). Outcomes included measures of CRF (ie, maximal oxygen consumption [VO2peak]) and energetic efficiency (ie, energetic cost of walking and RMR). RESULTS:After adjusting for demographics, comorbidities, and self-reported physical activity, longer WBL was associated with lower VO2peak (B= -1.01 ml/kg/min, P < .01) and higher RMR (B = 43.25 kcal, P < .05), lower SE was associated with lower VO2peak (B = 1.07 ml/kg/min, P < .01), and shorter TST was associated with lower VO2peak (B = 0.33 ml/kg/min, P < .05). Higher SE was associated with lower RMR among middle-aged adults but not older adults (interaction P-value < 0.05). CONCLUSION:Shorter TST, longer WBL, and lower SE are associated with poorer CRF and energetic efficiency among middle-aged and older adults. Longitudinal studies are needed to understand the temporality of these associations and potential targets for interventions in these populations.

BACKGROUND:Low vitamin D and B12 levels are linked to low self-reported sleep quality and conditions comorbid with sleep disturbances in older adults. We examined the associations between serum vitamin D and B12 levels with actigraphic sleep indices in middle-aged and older adults. METHODS:We studied 460 participants aged 40 and older [70.9±12.0(sd)years; 52.8% female; 22.2% Black] in the Baltimore Longitudinal Study of Aging. Predictors included total serum vitamin D and B12,sleep outcomes included total sleep time (minutes), sleep efficiency (%), sleep onset latency (minutes), wake after sleep onset (minutes), and average wake bout length (minutes). We examined race and sex as effect modifiers of associations between vitaminsand sleep. RESULTS:Higher vitamin D concentration was associated with longer total sleep time (B = 23.63 minutes, 95% CI: 9.62, 37.64) and shorter wake bout length (B = -0.29 minutes, 95% CI: -0.50, -0.09). Vitamin D deficiency was associated with shorter total sleep time (B=-23.03 minutes, 95% CI: -42.05 -4.01). The associations between vitamin D and sleep were stronger among Black participants. Higher B12 was associated with shorter wake after sleep onset in women but longer wake after sleep onset in men. B12 insufficiency/deficiency was associated with longer total sleep time in men but shorter total sleep time in women. CONCLUSIONS:Higher vitamin D levels are associated with longer and less disturbed sleep in middle-aged and older adults. Future studies are needed to understand if maintaining optimal vitamin D levels improve sleep quality and promote better sleep-associated health outcomes.

Study Objectives:Sleep and adipokines are important regulators of cardiometabolic health, yet their relationship in older adults remains poorly understood. We examined cross-sectional and longitudinal associations of actigraphic sleep parameters with adipokine levels among older adults and explored sex and central obesity as potential moderators. Materials and Methods:We studied 350 participants aged ≥60 years (mean = 74.8 ± 8.3 years, 48.9% females, 19.4% black) from the Baltimore Longitudinal Study of Aging who completed 6.5 ± 1.1 nights of wrist actigraphy and provided fasting plasma for adipokine measurements. Predictors were total sleep time (TST), sleep onset latency, wake after sleep onset, sleep efficiency, and average wake bout length (AWBL); outcomes were adiponectin and leptin levels. Results:At baseline, longer AWBL was associated with lower adiponectin (B = -2.02, 95% CI [-3.61, -0.42], p = .013) and leptin (B = -3.14, [-5.25, -1.03], p = .004) in fully adjusted models. Longer TST, modeled continuously, predicted a greater increase in adiponectin levels over time (B = 0.26, [0.081, 0.44], p = .007), and short TST (<6 h) was linked to subsequent decline in adiponectin (B = -0.90, [-1.72, -0.079], p = .038). Sex and central obesity moderated the baseline AWBL-adiponectin associations, which were significant in females (B = -3.92, [-6.25, -1.59], p = .001) and those with lower central obesity (B = -6.98, [-10.43, -3.53], p < .001). Conclusions:Longer sleep duration and shorter wake bouts are linked to more favorable adipokine profiles in older adults, especially in females and those with lower central obesity. Sleep may exert cardiometabolic effects through adipokines in older adults.

INTRODUCTION:Circadian rest/activity rhythms (RARs) change across adulthood, and alterations in RARs have been associated with cognitive decline, Alzheimer's disease (AD), and dementia. Little is known, however, about associations of RARs with subsequent changes in brain structure. METHODS:We investigated cross-sectional and longitudinal associations between RARs from wrist actigraphy and AD-relevant brain volumes from magnetic resonance imaging in cognitively unimpaired adults aged ≥ 50 and whether age moderated these associations. RESULTS:In cross-sectional analyses, weaker and more fragmented RARs were associated with smaller medial temporal region volumes. Longitudinally, more regular and less fragmented RARs were associated with better maintenance of amygdala volume over time. Further, associations between RAR fragmentation and global atrophy were strongest among the oldest participants. DISCUSSION:Results link RARs with medial temporal lobe and global brain volumes and indicate that these associations may differ by age. Research is needed on the effects of RAR-focused interventions on brain health.

Evidence regarding the potential adverse impact of sleep medications on hearing is inconsistent and limited. Potential mechanisms include decreased input from the auditory nerve and disturbed organization and integration of sensory information. This study aims to investigate the associations between trajectories of sleep medication use over nearly 30 years and late-life hearing among community-dwelling adults. Participants in the Atherosclerosis Risk in Communities (ARIC) Study underwent hearing evaluations at visit 6 (2016-17) and provided medications used in the past four weeks for each visit from visits 1 (1987-89) to 6. Hearing outcomes included: (1) Peripheral auditory function: better-ear four-frequency (0.5, 1, 2, 4 kHz) pure-tone average (PTA, higher=worse) assessed by pure-tone audiometry in decibels (dB); (2) Central auditory function: Quick Speech-in-noise (QuickSIN) test measures the ability to understand speech in noise, with score ranging from 0-30 (higher=better). Sleep medication included barbiturates, benzodiazepine derivatives, non-selective serotonin reuptake inhibitor antidepressants, and hypnotics. Sleep medication use was considered as any vs. none at each visit and group-based trajectory modeling identified three trajectory groups: non-users (N=2,398), increasing users (N=449), and continuous users (N=255). Linear regression models were used to estimate differences in hearing associated with sleep medication use trajectory groups. Models adjusted for demographics, lifestyle, and cardiovascular factors. We further conducted stratification analysis by visit 2 (1990-92) self-reported insomnia symptoms, including having trouble falling asleep and waking up repeatedly (yes vs. no). Among 3,102 participants (mean visit 1 age=50.7 years, 59% female, 21% Black), when compared to non-users, increasing users had 1.61 dB (95% confidence interval [CI]: 0.38, 2.84) significantly worse PTA; no differences were observed in QuickSIN. The association was significant among participants without trouble falling asleep (1.93 dB, 95% CI: 0.53, 3.34), but not among those with the symptom (0.53 dB, 95% CI: -2.22, 3.28). However, for waking up repeatedly, the association was stronger among those with the symptom (Yes: 2.35 dB, 95% CI: 0.41, 4.29; No: 1.29 dB, 95% CI: -0.36, 2.94). This study provides initial evidence linking sleep medication use from midlife to late life with worse late-life hearing, and the association might differ by sleep symptoms.

Weight loss medications are emerging candidates for pharmacotherapy of sleep-disordered breathing (SDB). A melanocortin 4 receptor (MC4R) agonist, setmelanotide (Set), is used to treat obesity caused by abnormal melanocortin and leptin signaling. We hypothesized that Set can treat SDB in mice with diet-induced obesity. We performed a proof-of-concept randomized crossover trial of a single dose of Set versus vehicle and a 2-week daily Set versus vehicle trial, examined colocalization of Mc4r mRNAs with the markers of CO2-sensing neurons Phox2b and neuromedin B in the brainstem, and expressed Cre-dependent designer receptors exclusively activated by designer drugs (DREADDs) or caspase in obese Mc4r-Cre mice. Set increased minute ventilation across sleep/wake states, enhanced the hypercapnic ventilatory response (HCVR), and abolished apneas during sleep. Phox2b+ neurons in the nucleus of the solitary tract (NTS) and the parafacial region expressed Mc4r. Chemogenetic stimulation of the MC4R+ neurons in the parafacial region, but not in the NTS, augmented HCVR without any changes in metabolism. Caspase elimination of the parafacial MC4R+ neurons abolished effects of Set on HCVR. Parafacial MC4R+ neurons projected to the respiratory premotor neurons retrogradely labeled from C3-C4. In conclusion, MC4R agonists enhance the HCVR and treat SDB by acting on the parafacial MC4R+ neurons.

Prolonged wakefulness leads to persistent, deep recovery sleep (RS). However, the neuronal circuits that mediate this process remain elusive. From a circuit screen in mice, we identified a group of thalamic nucleus reuniens (RE) neurons activated during sleep deprivation (SD) and required for sleep homeostasis. Optogenetic activation of RE neurons leads to an unusual phenotype: presleep behaviors (grooming and nest organizing) followed by prolonged, intense sleep that resembles RS. Inhibiting RE activity during SD impairs subsequent RS, which suggests that these neurons signal sleep need. RE neurons act upstream of sleep-promoting zona incerta cells, and SD triggers plasticity of this circuit to strengthen their connectivity. These findings reveal a circuit mechanism by which sleep need transforms the functional coupling of a sleep circuit to promote persistent, deep sleep.

The daily cycles of sleep and arousal are among the most prominent biological rhythms under circadian control. While the role of the suprachiasmatic nucleus (SCN) as the master circadian pacemaker is well-established, the molecular and circuit mechanisms by which it regulates the rhythms of sleep and arousal remain poorly understood. It has previously shown that the Drosophila clock-output molecule Wide Awake (WAKE) and its mammalian homolog mWAKE are expressed in fly clock neurons and dorsomedial hypothalamus neurons that promote arousal. Here, it is investigated whether mWAKE labels an arousal-promoting SCN subcircuit in mice. Broad chemogenetic activation and inhibition of mWAKE+ cells lead to a marked increase in wakefulness and a stupor-like phenotype, respectively. Optogenetic activation specifically of mWAKE-expressing neurons in the SCN (SCNmWAKE neurons) promotes arousal, while genetic knockout of mWAKE or impairment of CLOCK function in these neurons enhances wakefulness selectively at night. The latter phenotype corresponds to a specific increase in nighttime spiking of SCNmWAKE neurons in mWake mutants. At the circuit level, SCNmWAKE neuron signaling to the subparaventricular zone (SPZ) yields a stronger arousal phenotype. Together, these findings suggest that the genetic mechanisms regulating circadian control of sleep and arousal are evolutionarily conserved and define a clock-regulated neural network important for rhythmic arousal.

The central amygdala (CeA) is an important neuronal hub that integrates external sensory inputs and information about internal states to regulate a range of innate and learned behaviors, including fear learning and memory. Prior studies, leveraging robust fear conditioning assays, have delineated detailed circuit mechanisms underlying the acquisition and recall of fear memories. However, the specific molecular mechanisms underlying these processes in the CeA remain poorly understood. Here, we investigate the role of the clock output molecule mWAKE/ANKFN1 within the CeA of male mice in fear learning and memory. mWAKE is expressed in multiple neuronal subclusters in the lateral CeA. Surprisingly, mWAKE levels do not exhibit rhythmic expression in the CeA. In line with this observation, expression of the core clock genes PER2 and BMAL also does not cycle in the CeA. Consistent with prior studies, loss of mWAKE function increases intrinsic excitability of CeA neurons. Furthermore, conditional knock-out of mWAKE and chemogenetic activation of CeAmWAKE neurons impair fear learning and memory. Finally, we show that mWAKE levels in a subset of CeA neurons are reduced following fear conditioning. These findings suggest a potential molecular mechanism modulating the activity and function of CeA neurons in fear learning and memory.

There is great interest in using genetically tractable organisms such as Drosophila to gain insights into the regulation and function of sleep. However, sleep phenotyping in Drosophila has largely relied on simple measures of locomotor inactivity. Here, we present FlyVISTA, a machine learning platform to perform deep phenotyping of sleep in flies. This platform comprises a high-resolution closed-loop video imaging system, coupled with a deep learning network to annotate 35 body parts, and a computational pipeline to extract behaviors from high-dimensional data. FlyVISTA reveals the distinct spatiotemporal dynamics of sleep and wake-associated microbehaviors at baseline, following administration of the sleep-inducing drug gaboxadol, and with dorsal fan-shaped body drivers. We identify a microbehavior ("haltere switch") exclusively seen during quiescence that indicates a deeper sleep stage. These results enable the rigorous analysis of sleep in Drosophila and set the stage for computational analyses of microbehaviors in quiescent animals.

Sleep pressure, the accumulating drive towards sleep during wakefulness, is shaped by Lhx6-positive GABAergic neurons in the zona incerta (ZI). Here, we show that these neurons are broadly activated both by spontaneous and experimentally-elevated sleep pressure, and remain active for hours into recovery sleep. Activation is stronger in anterior ZI and varies across molecularly defined subgroups: Nkx2-2-positive neurons respond vigorously, whereas Calb2-positive neurons respond weakly. We also identify Lhx6-negative/Slc32a1-positive GABAergic ZI neurons with distinct sleep pressure responses. Selective genetic ablation of Nkx2-2 in Lhx6-positive neurons reduces the number of Lhx6-positive neurons, alters their distribution, blunts sleep pressure-induced activation of both Lhx6-positive and negative cells, and increases sleep duration. These findings show that developmentally specified, molecularly heterogeneous Lhx6-positive ZI neurons form a key hub for regulating sleep homeostasis, and offer new insight into the circuitry that controls sleep pressure.

We sought to explore whether genetic risk for, and self-reported, short sleep are associated with biological aging and whether age and sex moderate these associations. Participants were a subset of individuals from the Baltimore Longitudinal Study of Aging who had complete data on self-reported sleep (n = 567) or genotype (n = 367). Outcomes included: Intrinsic Horvath age, Hannum age, PhenoAge, GrimAge, and DNAm-based estimates of plasminogen activator inhibitor-1 (PAI-1) and granulocyte count. Results demonstrated that polygenic risk for short sleep was positively associated with granulocyte count; compared to those reporting <6 hr sleep, those reporting >7 hr demonstrated faster PhenoAge and GrimAge acceleration and higher estimated PAI-1. Polygenic risk for short sleep and self-reported sleep duration interacted with age and sex in their associations with some of the outcomes. Findings highlight that polygenic risk for short sleep and self-reported long sleep is associated with variation in the epigenetic landscape and subsequently aging.

BackgroundSleep problem is a common complication of Alzheimer’s disease (AD). Extensive preclinical studies have been performed to investigate the AD pathology. However, the pathophysiological consequence of AD complicated by sleep problem remains to be further determined.PurposeTo investigate brain metabolism and perfusion in an AD mouse model complicated by sleep problem, and subsequently identify potential imaging markers to better understand the associated pathophysiology.MethodsWe examined the oxygen extraction fraction (OEF), cerebral metabolic rate of oxygen (CMRO2), and cerebral blood flow (CBF) using state-of-the-art MRI techniques in a cohort of 5xFAD model mice. Additionally, neuroinflammation, indicated by activated microglia, was assessed using histology techniques. Sleep fragmentation (SF) was utilized as a representative for sleep problems.ResultsSF was associated with significant increases in OEF (P = 0.023) and CMRO2 (P = 0.029), indicating a state of hypermetabolism. CBF showed a significant genotype-by-sleep interaction effect (P = 0.026), particularly in the deep brain regions such as the hippocampus and thalamus. Neuroinflammation was primarily driven by genotype rather than SF, especially in regions with significant interaction effect in CBF measurements.ConclusionThese results suggest that brain metabolism and perfusion measurements are promising markers for studying the co-pathogenesis of AD and SF.

Sleep is regulated by homeostatic sleep drive and the circadian clock. While tremendous progress has been made in elucidating the molecular components of the core circadian oscillator, the output mechanisms by which this robust oscillator generates rhythmic sleep behavior remain poorly understood. At the cellular level, growing evidence suggests that subcircuits in the master circadian pacemaker suprachiasmatic nucleus (SCN) in mammals and in the clock network in Drosophila regulate distinct aspects of sleep. Thus, to identify novel molecules regulating the circadian timing of sleep, we conducted a large-scale screen of mouse SCN-enriched genes in Drosophila Here, we show that Tob (Transducer of ERB-B2) regulates the timing of sleep onset at night in female fruit flies. Knockdown of Tob pan-neuronally, either constitutively or conditionally, advances sleep onset at night. We show that Tob is specifically required in "evening neurons" (the LNds and the fifth s-LNv) of the clock network for proper timing of sleep onset. Tob levels cycle in a clock-dependent manner in these neurons. Silencing of these "evening" clock neurons results in an advanced sleep onset at night, similar to that seen with Tob knockdown. Finally, sharp intracellular recordings demonstrate that the amplitude and kinetics of LNd postsynaptic potentials (PSPs) cycle between day and night, and this cycling is attenuated with Tob knockdown in these cells. Our data suggest that Tob acts as a clock output molecule in a subset of clock neurons to potentiate their activity in the evening and enable the proper timing of sleep onset at night.

Animals exhibit rhythmic patterns of behavior that are shaped by an internal circadian clock and the external environment. While light intensity varies across the day, there are particularly robust differences at twilight (dawn/dusk). These periods are also associated with major changes in behavioral states, such as the transition from arousal to sleep. However, the neural mechanisms by which time and environmental conditions promote these behavioral transitions are poorly defined. Here, we show that the E1 subclass of Drosophila evening clock neurons promotes the transition from arousal to sleep at dusk. We first demonstrate that the cell-autonomous clocks of E2 neurons alone are required to drive and adjust the phase of evening anticipation, the canonical behavior associated with "evening" clock neurons. We next show that conditionally silencing E1 neurons causes a significant delay in sleep onset after dusk. However, rather than simply promoting sleep, activating E1 neurons produces time- and light- dependent effects on behavior. Activation of E1 neurons has no effect early in the day, but then triggers arousal before dusk and induces sleep after dusk. Strikingly, these phenotypes critically depend on the presence of light during the day. Despite their influence on behavior around dusk, in vivo voltage imaging of E1 neurons reveals that their spiking rate does not vary between dawn and dusk. Moreover, E1-specific clock ablation has no effect on arousal or sleep. Thus, we suggest that, rather than specifying "evening" time, E1 neurons act, in concert with other rhythmic neurons, to promote behavioral transitions at dusk.

Abstract Introduction Prolonged wakefulness leads to persistent, deep recovery sleep. However, the neuronal circuits mediating this process remain elusive. Here, starting from a large circuit screen, we identify and characterize a group of thalamic nucleus reuniens (RE) neurons activated by sleep pressure and required for sleep homeostasis. Methods To identify upstream excitatory neuronal populations inducing NREM sleep, we performed rabies virus-mediated screening in mice. Sleep/wake states were assayed using tethered EEG/EMG recordings. Activation or inhibition of neuronal activity was performed using chemogenetic and/or optogenetic techniques. Behavior during optogenetic RE activation was assessed using video recordings. To genetically access the sleep pressure-activated RE neurons, Targeted Recombination in Active Populations (TRAP2) mice were used. Viral expression of caspase3 was used for selective ablation of cells. For projection analyses, virally expressed Synaptophysin was used as a presynaptic marker. To visually examine synaptic connectivity, we performed enhanced Green fluorescent protein Reconstruction Across Synaptic Partners (eGRASP) analysis. Functional connectivity between RE to downstream neurons was tested by slice patch-clamp recordings. Results From a circuit screen in mice, we identified a group of excitatory thalamic RE neurons activated by sleep pressure and required for sleep homeostasis. Optogenetic activation of these neurons leads to an unusual phenotype: sleep-preparatory behaviors (e.g., nesting) followed by prolonged, intense sleep resembling recovery sleep. The activity of RE neurons (assessed by Fos) is increased by sleep deprivation (SD), and ablation of these Fos+ RE cells markedly impairs recovery sleep following SD. Moreover, inhibiting RE activity during SD impairs subsequent recovery sleep, suggesting that these neurons signal sleep need. RE neurons act upstream of sleep-promoting zona incerta (ZI) cells, and, remarkably, SD triggers plasticity of this circuit to strengthen its connectivity, as assessed by eGRASP analysis and patch-clamp recordings. Finally, our results indicate that CaMKII signaling is required for morphological plasticity of the RE-ZI circuit and persistence of homeostatic NREM sleep. Conclusion A subset of RE neurons encode sleep pressure and generate persistent NREM sleep, via a projection to sleep-promoting ZI neurons. Increased sleep need triggers plasticity of the RE-ZI projection which enhances functional connectivity facilitating the transmission of homeostatic sleep drive. Support (if any) NIH grants: R01NS094571, R35NS122181, S10OD023548, NS05027

Animal behavior depends on internal state. While subtle movements can signify significant changes in internal state, computational methods for analyzing these "microbehaviors" are lacking. Here, we present FlyVISTA, a machine-learning platform to characterize microbehaviors in freely-moving flies, which we use to perform deep phenotyping of sleep. This platform comprises a high-resolution closed-loop video imaging system, coupled with a deep-learning network to annotate 35 body parts, and a computational pipeline to extract behaviors from high-dimensional data. FlyVISTA reveals the distinct spatiotemporal dynamics of sleep-associated microbehaviors in flies. We further show that stimulation of dorsal fan-shaped body neurons induces micromovements, not sleep, whereas activating R5 ring neurons triggers rhythmic proboscis extension followed by persistent sleep. Importantly, we identify a novel microbehavior ("haltere switch") exclusively seen during quiescence that indicates a deeper sleep stage. These findings enable the rigorous analysis of sleep in Drosophila and set the stage for computational analyses of microbehaviors.

Circadian rhythms are generated by the master pacemaker suprachiasmatic nucleus (SCN) in concert with local clocks throughout the body. Although many brain regions exhibit cycling clock gene expression, the identity of a discrete extra-SCN brain oscillator that produces rhythmic behavior has remained elusive. Here, we show that an extra-SCN oscillator in the lateral amygdala (LA) is defined by expression of the clock-output molecule mWAKE/ANKFN1. mWAKE is enriched in the anterior/dorsal LA (adLA), and, strikingly, selective disruption of clock function or excitatory signaling in adLAmWAKE neurons abolishes Period2 (PER2) rhythms throughout the LA. mWAKE levels rise at night and promote rhythmic excitability of adLAmWAKE neurons by upregulating Ca2+-activated K+ channel activity specifically at night. adLAmWAKE neurons coordinate rhythmic sensory perception and anxiety in a clock-dependent and WAKE-dependent manner. Together, these data reveal the cellular identity of an extra-SCN brain oscillator and suggest a multi-level hierarchical system organizing molecular and behavioral rhythms.

STUDY OBJECTIVES:To compare sleep and 24-hour rest/activity rhythms (RARs) between cognitively normal older adults who are β-amyloid-positive (Aβ+) or Aβ- and replicate a novel time-of-day-specific difference between these groups identified in a previous exploratory study. METHODS:We studied 82 cognitively normal participants from the Baltimore Longitudinal Study of Aging (aged 75.7 ± 8.5 years, 55% female, 76% white) with wrist actigraphy data and Aβ+ versus Aβ- status measured by [11C] Pittsburgh compound B positron emission tomography. RARs were calculated using epoch-level activity count data from actigraphy. We used novel, data-driven function-on-scalar regression analyses and standard RAR metrics to cross-sectionally compare RARs between 25 Aβ+ and 57 Aβ- participants. RESULTS:Compared to Aβ- participants, Aβ+ participants had higher mean activity from 1:00 p.m. to 3:30 p.m. when using less conservative pointwise confidence intervals (CIs) and from 1:30 p.m. to 2:30 p.m. using more conservative, simultaneous CIs. Furthermore, Aβ+ participants had higher day-to-day variability in activity from 9:00 a.m. to 11:30 a.m. and lower variability from 1:30 p.m. to 4:00 p.m. and 7:30 p.m. to 10:30 p.m. according to pointwise CIs, and lower variability from 8:30 p.m. to 10:00 p.m. using simultaneous CIs. There were no Aβ-related differences in standard sleep or RAR metrics. CONCLUSIONS:Findings suggest Aβ+ older adults have higher, more stable day-to-day afternoon/evening activity than Aβ- older adults, potentially reflecting circadian dysfunction. Studies are needed to replicate our findings and determine whether these or other time-of-day-specific RAR features have utility as markers of preclinical Aβ deposition and if they predict clinical dementia and agitation in the afternoon/evening (i.e. "sundowning").