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The suprachiasmatic nucleus is the circadian pacemaker of the mammalian brain. Suprachiasmatic nucleus neurons display synchronization of their firing frequency on a circadian timescale, which is required for the pacemaker function of the suprachiasmatic nucleus. However, the mechanisms by which suprachiasmatic nucleus neurons remain synchronized in vivo are poorly understood, although synaptic communication is considered indispensable. Suprachiasmatic nucleus neurons contain the neurotransmitter GABA and express GABA receptors. This has inspired the hypothesis that GABA signalling may play a central role in network synchronization, although this remains untested in vivo. Here, using local genetic deletion, we show that disruption of GABA synaptic transmission within the suprachiasmatic nucleus of adult mice results in the eventual deterioration of physiological and behavioural rhythmicity in vivo and concomitant cellular desynchrony in vitro. These findings suggest that intercellular GABA signalling is essential for behavioural rhythmicity and cellular synchrony of the suprachiasmatic nucleus neural network., (© 2024 The Author(s). European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Tumor-associated macrophages (TAMs) are prime therapeutic targets due to their pro-tumorigenic functions, but varying efficacy of macrophage-targeting therapies highlights our incomplete understanding of how macrophages are regulated within the tumor microenvironment (TME). The circadian clock is a key regulator of macrophage function, but how circadian rhythms of macrophages are influenced by the TME remains unknown. Here, we show that conditions associated with the TME such as polarizing stimuli, acidic pH, and lactate can alter circadian rhythms in macrophages. While cyclic AMP (cAMP) has been reported to play a role in macrophage response to acidic pH, our results indicate pH-driven changes in circadian rhythms are not mediated solely by cAMP signaling. Remarkably, circadian disorder of TAMs was revealed by clock correlation distance analysis. Our data suggest that heterogeneity in circadian rhythms within the TAM population level may underlie this circadian disorder. Finally, we report that circadian regulation of macrophages suppresses tumor growth in a murine model of pancreatic cancer. Our work demonstrates a novel mechanism by which the TME influences macrophage biology through modulation of circadian rhythms., (© 2024. The Author(s).)

Macrophages are prime therapeutic targets due to their pro-tumorigenic and immunosuppressive functions in tumors, but the varying efficacy of therapeutic approaches targeting macrophages highlights our incomplete understanding of how the tumor microenvironment (TME) can influence regulation of macrophages. The circadian clock is a key internal regulator of macrophage function, but how circadian rhythms of macrophages may be influenced by the tumor microenvironment remains unknown. We found that conditions associated with the TME such as polarizing stimuli, acidic pH, and elevated lactate concentrations can each alter circadian rhythms in macrophages. Circadian rhythms were enhanced in pro-resolution macrophages but suppressed in pro-inflammatory macrophages, and acidic pH had divergent effects on circadian rhythms depending on macrophage phenotype. While cyclic AMP (cAMP) has been reported to play a role in macrophage response to acidic pH, our results indicate that pH-driven changes in circadian rhythms are not mediated solely by the cAMP signaling pathway. Remarkably, clock correlation distance analysis of tumor-associated macrophages (TAMs) revealed evidence of circadian disorder in TAMs. This is the first report providing evidence that circadian rhythms of macrophages are altered within the TME. Our data further suggest that heterogeneity in circadian rhythms at the population level may underlie this circadian disorder. Finally, we sought to determine how circadian regulation of macrophages impacts tumorigenesis, and found that tumor growth was suppressed when macrophages had a functional circadian clock. Our work demonstrates a novel mechanism by which the tumor microenvironment can influence macrophage biology through altering circadian rhythms, and the contribution of circadian rhythms in macrophages to suppressing tumor growth.

The brain is a complex organ, fundamentally changing across the day to perform basic functions like sleep, thought, and regulating whole-body physiology. This requires a complex symphony of nutrients, hormones, ions, neurotransmitters and more to be properly distributed across the brain to maintain homeostasis throughout 24 hours. These solutes are distributed both by the blood and by cerebrospinal fluid. Cerebrospinal fluid contents are distinct from the general circulation because of regulation at brain barriers including the choroid plexus, glymphatic system, and blood-brain barrier. In this review, we discuss the overlapping circadian (≈24-hour) rhythms in brain fluid biology and at the brain barriers. Our goal is for the reader to gain both a fundamental understanding of brain barriers alongside an understanding of the interactions between these fluids and the circadian timing system. Ultimately, this review will provide new insight into how alterations in these finely tuned clocks may lead to pathology., Competing Interests: None.

Functional hyperemia, also known as neurovascular coupling, is a phenomenon that occurs when neural activity increases local cerebral blood flow. Because all biological activity produces metabolic waste, we here sought to investigate the relationship between functional hyperemia and waste clearance via the glymphatic system. The analysis showed that whisker stimulation increased both glymphatic influx and clearance in the mouse somatosensory cortex with a 1.6-fold increase in periarterial cerebrospinal fluid (CSF) influx velocity in the activated hemisphere. Particle tracking velocimetry revealed a direct coupling between arterial dilation/constriction and periarterial CSF flow velocity. Optogenetic manipulation of vascular smooth muscle cells enhanced glymphatic influx in the absence of neural activation. We propose that impedance pumping allows arterial pulsatility to drive CSF in the same direction as blood flow, and we present a simulation that supports this idea. Thus, functional hyperemia boosts not only the supply of metabolites but also the removal of metabolic waste., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The glymphatic system is a fluid transport network of cerebrospinal fluid (CSF) entering the brain along arterial perivascular spaces, exchanging with interstitial fluid (ISF), ultimately establishing directional clearance of interstitial solutes. CSF transport is facilitated by the expression of aquaporin-4 (AQP4) water channels on the perivascular endfeet of astrocytes. Mice with genetic deletion of AQP4 (AQP4 KO) exhibit abnormalities in the brain structure and molecular water transport. Yet, no studies have systematically examined how these abnormalities in structure and water transport correlate with glymphatic function. Here, we used high-resolution 3D magnetic resonance (MR) non-contrast cisternography, diffusion-weighted MR imaging (MR-DWI) along with intravoxel-incoherent motion (IVIM) DWI, while evaluating glymphatic function using a standard dynamic contrast-enhanced MR imaging to better understand how water transport and glymphatic function is disrupted after genetic deletion of AQP4. AQP4 KO mice had larger interstitial spaces and total brain volumes resulting in higher water content and reduced CSF space volumes, despite similar CSF production rates and vascular density compared to wildtype mice. The larger interstitial fluid volume likely resulted in increased slow but not fast MR diffusion measures and coincided with reduced glymphatic influx. This markedly altered brain fluid transport in AQP4 KO mice may result from a reduction in glymphatic clearance, leading to enlargement and stagnation of fluid in the interstitial space. Overall, diffusion MR is a useful tool to evaluate glymphatic function and may serve as valuable translational biomarker to study glymphatics in human disease., Competing Interests: RG, LH, HM, MG, TD, NH, LX, WP, PM, MN, YM No competing interests declared, (© 2023, Gomolka et al.)

Glymphatic fluid transport eliminates metabolic waste from the brain including amyloid-β, yet the methodology for studying efflux remains rudimentary. Here, we develop a method to evaluate glymphatic real-time clearance. Efflux of Direct Blue 53 (DB53, also T-1824 or Evans Blue) injected into the striatum is quantified by imaging the DB53 signal in the vascular compartment, where it is retained due to its high affinity to albumin. The DB53 signal is detectable as early as 15 min after injection and the efflux kinetics are sharply reduced in mice lacking the water channel aquaporin 4 (AQP4). Pharmacokinetic modeling reveal that DB53 efflux is consistent with the existence of two efflux paths, one with fast kinetics (T 1/2  = 50 min) and another with slow kinetics (T 1/2  = 240 min), in wild-type mice. This in vivo methodology will aid in defining the physiological variables that drive efflux, as well as the impact of brain states or disorders on clearance kinetics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Inc.)

All biological processes including movement, digestion, and thought require energy, and the metabolic activity necessary to generate that energy also generates waste. Metabolic by-products, including a variety of proteins, accumulate in the tissue and can be harmful if not removed. Throughout the body, the lymphatic system is responsible for clearing metabolic waste from the tissue (Figure 1). An ultrafiltrate of blood plasma constantly enters the tissue from the microvasculature and percolates through the tissue before uptake by lymphatic capillaries. Once in these capillaries, a series of lymphatic vessels, pumps, and valves ensures directional fluid movement, ultimately draining the lymph containing waste products back into the venous circulatory system. The liver can be regarded as the ultimate recycling station in the body, where these waste products are either degraded or reused., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Throughout the body, lymphatic fluid movement supports critical functions including clearance of excess fluid and metabolic waste. The glymphatic system is the analog of the lymphatic system in the CNS. As such, the glymphatic system plays a key role in regulating directional interstitial fluid movement, waste clearance, and, potentially, brain immunity. The glymphatic system enables bulk movement of CSF from the subarachnoid space along periarterial spaces, where it mixes with interstitial fluid within the parenchyma before ultimately exiting from the parenchyma via perivenous spaces. This review focuses on important questions about the structure of this system, why the brain needs a fluid transport system, and unexplored aspects of brain fluid transport. We provide evidence that astrocytes and blood vessels determine the shape of the perivascular space, ultimately controlling the movement of perivascular fluid. Glymphatic fluid movement has the potential to alter local as well as global transport of signaling molecules and metabolites. We also highlight the evidence for cross talk among the glymphatic system, cardiovascular system, gastrointestinal tract, and lymphatic system. Much remains to be studied, but we propose that the glymphatic/lymphatic system acts as a cornerstone in signaling between the brain and body., (Copyright © 2021 the authors.)

The glymphatic system is network of perivascular spaces through which cerebrospinal fluid and interstitial fluid can move through the brain, clearing metabolic waste, such as amyloid beta, lactate and more, from the parenchyma. This cleaning system is regulated by sleep and norepinephrine, with increased levels of norepinephrine during wakefulness inhibiting fluid movement. Norepinephrine is also essential for transition from acute to chronic pain, and sufferers of chronic neuropathic pain frequently present with sleep disruption. These connections among glymphatic clearance, sleep, and pain are very intriguing, and might lead to nonpharmaceutical interventions for pain treatment. This short perspective provides a rationale for the hypothesis that mind-body interventions-such as acupuncture-can reduce norepinephrine and increase glymphatic function, ultimately relieving chronic neuropathic pain., Competing Interests: No financial conflicts of interest exist., (Copyright 2020, Mary Ann Liebert, Inc., publishers.)

Sexual dimorphism is evident in brain structure, size, and function throughout multiple species. Here, we tested whether cerebrospinal fluid entry into the glymphatic system, a network of perivascular fluid transport that clears metabolic waste from the brain, was altered between male and female mice. We analyze glymphatic influx in 244 young reproductive age (2-4 months) C57BL/6 mice. We found no male/female differences in total influx under anesthesia, or across the anterior/posterior axis of the brain. Circadian-dependent changes in glymphatic influx under ketamine/xylazine anesthesia were not altered by sex. This was not true for diurnal rhythms under pentobarbital and avertin, but both still showed daily oscillations independent of biological sex. Finally, although glymphatic influx decreases with age there was no sex difference in total influx or subregion-dependent tracer distribution in 17 middle aged (9-10 months) and 36 old (22-24 months) mice. Overall, in healthy adult C57BL/6 mice we could not detect male/female differences in glymphatic influx. This finding contrasts the gender differences in common neurodegenerative diseases. We propose that additional sex-dependent co-morbidities, such as chronic stress, protein misfolding, traumatic brain injury or other pathological mechanisms may explain the increased risk for developing proteinopathies rather than pre-existing suppression of glymphatic influx.

The glymphatic system is a network of perivascular spaces that promotes movement of cerebrospinal fluid (CSF) into the brain and clearance of metabolic waste. This fluid transport system is supported by the water channel aquaporin-4 (AQP4) localized to vascular endfeet of astrocytes. The glymphatic system is more effective during sleep, but whether sleep timing promotes glymphatic function remains unknown. We here show glymphatic influx and clearance exhibit endogenous, circadian rhythms peaking during the mid-rest phase of mice. Drainage of CSF from the cisterna magna to the lymph nodes exhibits daily variation opposite to glymphatic influx, suggesting distribution of CSF throughout the animal depends on time-of-day. The perivascular polarization of AQP4 is highest during the rest phase and loss of AQP4 eliminates the day-night difference in both glymphatic influx and drainage to the lymph nodes. We conclude that CSF distribution is under circadian control and that AQP4 supports this rhythm.

The hypothalamic suprachiasmatic (SCN) clock contains several neurochemically defined cell groups that contribute to the genesis of circadian rhythms. Using cell-specific and genetically targeted approaches we have confirmed an indispensable role for vasoactive intestinal polypeptide-expressing SCN (SCN VIP ) neurons, including their molecular clock, in generating the mammalian locomotor activity (LMA) circadian rhythm. Optogenetic-assisted circuit mapping revealed functional, di-synaptic connectivity between SCN VIP neurons and dorsomedial hypothalamic neurons, providing a circuit substrate by which SCN VIP neurons may regulate LMA rhythms. In vivo photometry revealed that while SCN VIP neurons are acutely responsive to light, their activity is otherwise behavioral state invariant. Single-nuclei RNA-sequencing revealed that SCN VIP neurons comprise two transcriptionally distinct subtypes, including putative pacemaker and non-pacemaker populations. Altogether, our work establishes necessity of SCN VIP neurons for the LMA circadian rhythm, elucidates organization of circadian outflow from and modulatory input to SCN VIP cells, and demonstrates a subpopulation-level molecular heterogeneity that suggests distinct functions for specific SCN VIP subtypes.

Circadian rhythms are 24-h cycles in physiology regulated by the suprachiasmatic nucleus (SCN) in the brain, where daily cues act on SCN neurons to alter clock timing. Cannabinoid signaling modulates SCN neuronal activity, although the mechanism remains unclear. We propose that neuronal activity generates endocannabinoid release, activating astrocyte Ca 2+ signaling, which releases adenosine and activates adenosine-1 receptors (A1Rs) on the presynaptic axon terminals, decreasing GABA release. We demonstrated, in mice, that activation of cannabinoid-1 receptors (CB1R) with the agonist WIN 55,212-2 (WIN) reduced the miniature GABA receptor-mediated postsynaptic current (mGPSC) frequency by a mechanism that requires astrocytes and A1R. WIN activated an intracellular Ca 2+ signaling pathway in astrocytes. Activating this intracellular Ca 2+ pathway with designer receptors exclusively activated by designer drugs (DREADDs) also decreased the mGPSC frequency and required A1R activation. The frequency of spontaneous Ca 2+ events, including those induced by depolarization of a postsynaptic SCN neuron, was reduced by blocking CB1R activation with AM251, demonstrating neuronal endocannabinoid signaling modulates astrocytic Ca 2+ signaling in the SCN. Finally, daytime application of WIN or adenosine phase advanced the molecular circadian clock, indicating that this cannabinoid signaling pathway is vital for the timing of circadian rhythms., (Copyright © 2020 Hablitz et al.)

The glymphatic system is responsible for brain-wide delivery of nutrients and clearance of waste via influx of cerebrospinal fluid (CSF) alongside perivascular spaces and through the brain. Glymphatic system activity increases during sleep or ketamine/xylazine (K/X) anesthesia, yet the mechanism(s) facilitating CSF influx are poorly understood. Here, we correlated influx of a CSF tracer into the brain with electroencephalogram (EEG) power, heart rate, blood pressure, and respiratory rate in wild-type mice under six different anesthesia regimens. We found that glymphatic CSF tracer influx was highest under K/X followed by isoflurane (ISO) supplemented with dexmedetomidine and pentobarbital. Mice anesthetized with α-chloralose, Avertin, or ISO exhibited low CSF tracer influx. This is the first study to show that glymphatic influx correlates positively with cortical delta power in EEG recordings and negatively with beta power and heart rate.

The glymphatic system is a brain-wide clearance pathway; its impairment contributes to the accumulation of amyloid-β. Influx of cerebrospinal fluid (CSF) depends upon the expression and perivascular localization of the astroglial water channel aquaporin-4 (AQP4). Prompted by a recent failure to find an effect of Aqp4 knock-out (KO) on CSF and interstitial fluid (ISF) tracer transport, five groups re-examined the importance of AQP4 in glymphatic transport. We concur that CSF influx is higher in wild-type mice than in four different Aqp4 KO lines and in one line that lacks perivascular AQP4 ( Snta1 KO). Meta-analysis of all studies demonstrated a significant decrease in tracer transport in KO mice and rats compared to controls. Meta-regression indicated that anesthesia, age, and tracer delivery explain the opposing results. We also report that intrastriatal injections suppress glymphatic function. This validates the role of AQP4 and shows that glymphatic studies must avoid the use of invasive procedures., Competing Interests: HM, LH, AX, WF, WZ, TP, HM, GM, RC, MS, MP, VP, TD, BK, XW, BP, AT, IL, YA, MY, JT, MX, HH, AA, JI, MN No competing interests declared, (© 2018, Mestre et al.)

Attention-deficit/hyperactivity disorder (ADHD) is a common condition with comorbid insomnia reported in >70% of children and adults. These patients demonstrate delays in sleep-wake rhythms, nocturnal rise in melatonin, and early morning rise in cortisol. Given that standard psychopharmacologic treatments for ADHD often do not completely control symptoms in participants with circadian rhythm delay, we sought to test whether bright light therapy (BLT) advances circadian rhythms and further reduces ADHD symptoms over standard treatments. In addition to standard of care, participants with ADHD diagnosis underwent 1 week of baseline assessment followed by 2-weeks of 30-min morning 10,000-lux BLT beginning 3 h after mid-sleep time. Participants minimized overhead light after 4 p.m., wore an actigraphy watch, and recorded BLT time on daily sleep logs. Dim Light Melatonin Onset (DLMO) was assessed at baseline and after 2-week treatment. ADHD symptoms were measured by the ADHD-Rating Scales (ADHD-RS). BLT significantly advanced the phase of DLMO by 31 min [mean time (SEM), 20:36 (0:21) advanced to 20:05 (0:20)] and mid-sleep time by 57 min [4:37 (0:22) advanced to 3:40 (0:16); paired t-tests, p = 0.002 and 0.004, respectively). Phase advances (in DLMO or mid-sleep time) were significantly correlated with decreased ADHD-RS total scores (p = 0.027 and 0.044) and Hyperactive-Impulsive sub-scores (p = 0.014 and 0.013, respectively). Actigraphy analysis for a subset of 8 participants with significant DLMO phase advance revealed no significant changes in total sleep time, sleep efficiency, wake after sleep onset, or percent wake during sleep interval. This is the first successful use of BLT for advancing melatonin phase and improving ADHD symptoms in adults. BLT may be a complementary treatment for both delayed sleep timing and ADHD symptoms in adults., (Published by Elsevier Ltd.)

Hippocampal rhythms in clock gene expression, enzymatic activity, and long-term potentiation (LTP) are thought to underlie day-night differences in memory acquisition and recall. Glycogen synthase kinase 3-beta (GSK3β) is a known regulator of hippocampal function, and inhibitory phosphorylation of GSK3β exhibits region-specific differences over the light-dark cycle. Here, we sought to determine whether phosphorylation of both GSK3α and GSK3β isoforms has an endogenous circadian rhythm in specific areas of the hippocampus and whether chronic inhibition or activation alters the molecular clock and hippocampal plasticity (LTP). Results indicated a significant endogenous circadian rhythm in phosphorylation of GSK3β, but not GSK3α, in hippocampal CA1 extracts from mice housed in constant darkness for at least 2 weeks. To examine the importance of this rhythm, genetic and pharmacological strategies were used to disrupt the GSK3 activity rhythm by chronically activating or inhibiting GSK3. Chronic activation of both GSK3 isoforms in transgenic mice (GSK3-KI mice) diminished rhythmic BMAL1 expression. On the other hand, chronic treatment with a GSK3 inhibitor significantly shortened the molecular clock period of organotypic hippocampal PER2::LUC cultures. While WT mice exhibited higher LTP magnitude at night compared to day, the day-night difference in LTP magnitude remained with greater magnitude at both times of day in mice with chronic GSK3 activity. On the other hand, pharmacological GSK3 inhibition impaired day-night differences in LTP by blocking LTP selectively at night. Taken together, these results support the model that circadian rhythmicity of hippocampal GSK3β activation state regulates day/night differences in molecular clock periodicity and a major form of synaptic plasticity (LTP)., (© 2017 Wiley Periodicals, Inc.)

Melatonin supplementation has been used as a therapeutic agent for several diseases, yet little is known about the underlying mechanisms by which melatonin synchronizes circadian rhythms. G-protein signaling plays a large role in melatonin-induced phase shifts of locomotor behavior and melatonin receptors activate G-protein-coupled inwardly rectifying potassium (GIRK) channels in Xenopus oocytes. The present study tested the hypothesis that melatonin influences circadian phase and electrical activity within the central clock in the suprachiasmatic nucleus (SCN) through GIRK channel activation. Unlike wild-type littermates, GIRK2 knock-out (KO) mice failed to phase advance wheel-running behavior in response to 3 d subcutaneous injections of melatonin in the late day. Moreover, in vitro phase resetting of the SCN circadian clock by melatonin was blocked by coadministration of a GIRK channel antagonist tertiapin-q (TPQ). Loose-patch electrophysiological recordings of SCN neurons revealed a significant reduction in the average action potential rate in response to melatonin. This effect was lost in SCN slices treated with TPQ and SCN slices from GIRK2 KO mice. The melatonin-induced suppression of firing rate corresponded with an increased inward current that was blocked by TPQ. Finally, application of ramelteon, a potent melatonin receptor agonist, significantly decreased firing rate and increased inward current within SCN neurons in a GIRK-dependent manner. These results are the first to show that GIRK channels are necessary for the effects of melatonin and ramelteon within the SCN. This study suggests that GIRK channels may be an alternative therapeutic target for diseases with evidence of circadian disruption, including aberrant melatonin signaling., Significance Statement: Despite the widespread use of melatonin supplementation for the treatment of sleep disruption and other neurological diseases such as epilepsy and depression, no studies have elucidated the molecular mechanisms linking melatonin-induced changes in neuronal activity to its therapeutic effects. Here, we used behavioral and electrophysiological techniques to address this scientific gap. Our results show that melatonin and ramelteon, a potent and clinically relevant melatonin receptor agonist, significantly affect the neurophysiological function of suprachiasmatic nucleus neurons through activation of G-protein-coupled inwardly rectifying potassium (GIRK) channels. Given the importance of GIRK channels for neuronal excitability (with >600 publications on these channels to date), our study should generate broad interest from neuroscientists in fields such as epilepsy, addiction, and cognition., (Copyright © 2015 the authors 0270-6474/15/3514957-09$15.00/0.)

G protein signalling within the central circadian oscillator, the suprachiasmatic nucleus (SCN), is essential for conveying time-of-day information. We sought to determine whether G protein-coupled inwardly rectifying potassium channels (GIRKs) modulate SCN physiology and circadian behaviour. We show that GIRK current and GIRK2 protein expression are greater during the day. Pharmacological inhibition of GIRKs and genetic loss of GIRK2 depolarized the day-time resting membrane potential of SCN neurons compared to controls. Behaviourally, GIRK2 knockout (KO) mice failed to shorten free running period in response to wheel access in constant darkness and entrained more rapidly to a 6 h advance of a 12 h:12 h light-dark (LD) cycle than wild-type (WT) littermate controls. We next examined whether these effects were due to disrupted signalling of neuropeptide Y (NPY), which is known to mediate non-photic phase shifts, attenuate photic phase shifts and activate GIRKs. Indeed, GIRK2 KO SCN slices had significantly fewer silent cells in response to NPY, likely contributing to the absence of NPY-induced phase advances of PER2::LUC rhythms in organotypic SCN cultures from GIRK2 KO mice. Finally, GIRK channel activation is sufficient to cause a non-photic-like phase advance of PER2::LUC rhythms on a Per2(Luc+/-) background. These results suggest that rhythmic regulation of GIRK2 protein and channel function in the SCN contributes to day-time resting membrane potential, providing a mechanism for the fine tuning responses to non-photic and photic stimuli. Further investigation could provide insight into disorders with circadian disruption comorbidities such as epilepsy and addiction, in which GIRK channels have been implicated., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

Endogenous circadian rhythms are entrained to the 24-h light/dark cycle by both light and nonphotic stimuli. During the day, nonphotic stimuli, such as novel wheel-induced exercise, produce large phase advances. Neuropeptide Y (NPY) release from the thalamus onto suprachiasmatic nucleus (SCN) neurons at least partially mediates this nonphotic signal. The authors examined the hypothesis that NPY-induced phase advances are accompanied by suppression of PER2 and are mediated by long-term depression of neuronal excitability in a phase-specific manner. First, it was found that NPY-induced phase advances in PER2::LUC SCN cultures are largest when NPY (2.35 µM) is given in the early part of the day (circadian time [CT] 0-6). In addition, PER2::LUC levels in NPY-treated (compared to vehicle-treated) samples were suppressed beginning 6-7 h after treatment. Similar NPY application to organotypic Per1::GFP SCN cultures resulted in long-term suppression of spike rate of green fluorescent protein-positive (GFP+) cells when slices were treated with NPY during the early or middle of the day (zeitgeber time [ZT] 2 or 6), but not during the late day (ZT 10). Furthermore, 1-h bath application of NPY to acute SCN brain slices decreased general neuronal activity measured through extracellular recordings. Finally, NPY-induced phase advances of PER2::LUC rhythms were blocked by latent depolarization with 34.5 mM K(+) 3 h after NPY application. These results suggest that NPY-induced phase advances may be mediated by long-term depression of neuronal excitability. This model is consistent with findings in other brain regions that NPY-induced persistent hyperpolarization underlies mechanisms of energy homeostasis, anxiety-related behavior, and thalamocortical synchronous firing.