Neuronal innervation regulates the secretion of neurotrophic myokines and exosomes from skeletal muscle.

Myokines and exosomes, originating from skeletal muscle, are shown to play a significant role in maintaining brain homeostasis. While exercise has been reported to promote muscle secretion, little is known about the effects of neuronal innervation and activity on the yield and molecular composition of biologically active molecules from muscle. As neuromuscular diseases and disabilities associated with denervation impact muscle metabolism, we hypothesize that neuronal innervation and firing may play a pivotal role in regulating secretion activities of skeletal muscles. We examined this hypothesis using an engineered neuromuscular tissue model consisting of skeletal muscles innervated by motor neurons. The innervated muscles displayed elevated expression of mRNAs encoding neurotrophic myokines, such as interleukin-6, brain-derived neurotrophic factor, and FDNC5, as well as the mRNA of peroxisome-proliferator-activated receptor γ coactivator 1α, a key regulator of muscle metabolism. Upon glutamate stimulation, the innervated muscles secreted higher levels of irisin and exosomes containing more diverse neurotrophic microRNAs than neuron-free muscles. Consequently, biological factors secreted by innervated muscles enhanced branching, axonal transport, and, ultimately, spontaneous network activities of primary hippocampal neurons in vitro. Overall, these results reveal the importance of neuronal innervation in modulating muscle-derived factors that promote neuronal function and suggest that the engineered neuromuscular tissue model holds significant promise as a platform for producing neurotrophic molecules.

Development of circadian neurovascular function and its implications.

The neurovascular system forms the interface between the tissue of the central nervous system (CNS) and circulating blood. It plays a critical role in regulating movement of ions, small molecules, and cellular regulators into and out of brain tissue and in sustaining brain health. The neurovascular unit (NVU), the cells that form the structural and functional link between cells of the brain and the vasculature, maintains the blood-brain interface (BBI), controls cerebral blood flow, and surveils for injury. The neurovascular system is dynamic; it undergoes tight regulation of biochemical and cellular interactions to balance and support brain function. Development of an intrinsic circadian clock enables the NVU to anticipate rhythmic changes in brain activity and body physiology that occur over the day-night cycle. The development of circadian neurovascular function involves multiple cell types. We address the functional aspects of the circadian clock in the components of the NVU and their effects in regulating neurovascular physiology, including BBI permeability, cerebral blood flow, and inflammation. Disrupting the circadian clock impairs a number of physiological processes associated with the NVU, many of which are correlated with an increased risk of dysfunction and disease. Consequently, understanding the cell biology and physiology of the NVU is critical to diminishing consequences of impaired neurovascular function, including cerebral bleeding and neurodegeneration.

Electrophysiology of the Suprachiasmatic Nucleus: Single-Unit Recording.

Oscillatory output from the suprachiasmatic nuclei (SCN) of the hypothalamus communicates time-of-day information to the brain and body. The SCN's intrinsic ~24-h rhythm can be measured in the neuronal firing rate both in vivo and in vitro, where it continues unperturbed. This robust reporter of endogenous physiology in the SCN brain slice can be widely used to study dynamic changes in SCN physiology, its changing sensitivity to phase-altering signals, and underlying mechanisms. To provide relevant and reproducible data, care must be taken to ensure health of the SCN brain slice. The methods detailed here have been proven to produce healthy, long-lived brain slices.

Tubulin acetylation promotes penetrative capacity of cells undergoing radial intercalation.

Post-translational modification of tubulin provides differential functions to microtubule networks. Here, we address the role of tubulin acetylation on the penetrative capacity of cells undergoing radial intercalation, which is the process by which cells move apically, insert between outer cells, and join an epithelium. There are opposing forces that regulate intercalation, namely, the restrictive forces of the epithelial barrier versus the penetrative forces of the intercalating cell. Positively and negatively modulating tubulin acetylation in intercalating cells alters the developmental timing such that cells with more acetylation penetrate faster. We find that intercalating cells preferentially penetrate higher-order vertices rather than the more prevalent tricellular vertices. Differential timing in the ability of cells to penetrate different vertices reveals that lower-order vertices represent more restrictive sites of insertion. We shift the accessibility of intercalating cells toward more restrictive junctions by increasing tubulin acetylation, and we provide a geometric-based mathematical model that describes our results.

Pituitary Adenylate Cyclase-Activating Peptide (PACAP)-Glutamate Co-transmission Drives Circadian Phase-Advancing Responses to Intrinsically Photosensitive Retinal Ganglion Cell Projections by Suprachiasmatic Nucleus.

Results from a variety of sources indicate a role for pituitary adenylate cyclase-activating polypeptide (PACAP) in light/glutamate-induced phase resetting of the circadian clock mediated by the retinohypothalamic tract (RHT). Attempts to block or remove PACAP's contribution to clock-resetting have generated phenotypes that differ in their responses to light or glutamate. For example, previous studies of circadian behaviors found that period-maintenance and early-night phase delays are intact in PACAP-null mice, yet there is a consistent deficit in behavioral phase-resetting to light stimulation in the late night. Here we report rodent stimulus-response characteristics of PACAP release from the RHT, and map these to responses of the suprachiasmatic nucleus (SCN) in intact and PACAP-deficient mouse hypothalamus with regard to phase-resetting. SCN of PACAP-null mice exhibit normal circadian rhythms in neuronal activity, but are "blind" to glutamate stimulating phase-advance responses in late night, although not in early night, consistent with previously reported selective lack of late-night light behavioral responsiveness of these mice. Induction of CREB phosphorylation, a hallmark of the light/glutamate response of the SCN, also is absent in SCN-containing ex vivo slices from PACAP-deficient mouse hypothalamus. PACAP replacement to the SCN of PACAP-null mice restored wild-type phase-shifting of firing-rate patterns in response to glutamate applied to the SCN in late night. Likewise, ex vivo SCN of wild-type mice post-orbital enucleation are unresponsive to glutamate unless PACAP also is restored. Furthermore, we demonstrate that the period of efficacy of PACAP at SCN nerve terminals corresponds to waxing of PACAP mRNA expression in ipRGCs during the night, and waning during the day. These results validate the use of PACAP-deficient mice in defining the role and specificity of PACAP as a co-transmitter with glutamate in ipRGC-RHT projections to SCN in phase advancing the SCN circadian rhythm in late night.

Multimodal Chemical Analysis of the Brain by High Mass Resolution Mass Spectrometry and Infrared Spectroscopic Imaging.

The brain functions through chemical interactions between many different cell types, including neurons and glia. Acquiring comprehensive information on complex, heterogeneous systems requires multiple analytical tools, each of which have unique chemical specificity and spatial resolution. Multimodal imaging generates complementary chemical information via spatially localized molecular maps, ideally from the same sample, but requires method enhancements that span from data acquisition to interpretation. We devised a protocol for performing matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance-mass spectrometry imaging (MSI), followed by infrared (IR) spectroscopic imaging on the same specimen. Multimodal measurements from the same tissue provide precise spatial alignment between modalities, enabling more advanced image processing such as image fusion and sharpening. Performing MSI first produces higher quality data from each technique compared to performing IR imaging before MSI. The difference is likely due to fixing the tissue section during MALDI matrix removal, thereby preventing analyte degradation occurring during IR imaging from an unfixed specimen. Leveraging the unique capabilities of each modality, we utilized pan sharpening of MS (mass spectrometry) ion images with selected bands from IR spectroscopy and midlevel data fusion. In comparison to sharpening with histological images, pan sharpening can employ a plethora of IR bands, producing sharpened MS images while retaining the fidelity of the initial ion images. Using Laplacian pyramid sharpening, we determine the localization of several lipids present within the hippocampus with high mass accuracy at 5 µm pixel widths. Further, through midlevel data fusion of the imaging data sets combined with k-means clustering, the combined data set discriminates between additional anatomical structures unrecognized by the individual imaging approaches. Significant differences between molecular ion abundances are detected between relevant structures within the hippocampus, such as the CA1 and CA3 regions. Our methodology provides high quality multiplex and multimodal chemical imaging of the same tissue sample, enabling more advanced data processing and analysis routines.

Functional Peptidomics: Stimulus- and Time-of-Day-Specific Peptide Release in the Mammalian Circadian Clock.

Daily oscillations of brain and body states are under complex temporal modulation by environmental light and the hypothalamic suprachiasmatic nucleus (SCN), the master circadian clock. To better understand mediators of differential temporal modulation, we characterize neuropeptide releasate profiles by nonselective capture of secreted neuropeptides in an optic nerve horizontal SCN brain slice model. Releasates are collected following electrophysiological stimulation of the optic nerve/retinohypothalamic tract under conditions that alter the phase of the SCN activity state. Secreted neuropeptides are identified by intact mass via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We found time-of-day-specific suites of peptides released downstream of optic nerve stimulation. Peptide release was modified differentially with respect to time-of-day by stimulus parameters and by inhibitors of glutamatergic or PACAPergic neurotransmission. The results suggest that SCN physiology is modulated by differential peptide release of both known and unexpected peptides that communicate time-of-day-specific photic signals via previously unreported neuropeptide signatures.

CLAMP/Spef1 regulates planar cell polarity signaling and asymmetric microtubule accumulation in the Xenopus ciliated epithelia.

Most epithelial cells polarize along the axis of the tissue, a feature known as planar cell polarity (PCP). The initiation of PCP requires cell-cell signaling via the noncanonical Wnt/PCP pathway. Additionally, changes in the cytoskeleton both facilitate and reflect this polarity. We have identified CLAMP/Spef1 as a novel regulator of PCP signaling. In addition to decorating microtubules (MTs) and the ciliary rootlet, a pool of CLAMP localizes at the apical cell cortex. Depletion of CLAMP leads to the loss of PCP protein asymmetry, defects in cilia polarity, and defects in the angle of cell division. Additionally, depletion of CLAMP leads to a loss of the atypical cadherin-like molecule Celrs2, suggesting that CLAMP facilitates the stabilization of junctional interactions responsible for proper PCP protein localization. Depletion of CLAMP also affects the polarized organization of MTs. We hypothesize that CLAMP facilitates the establishment of cell polarity and promotes the asymmetric accumulation of MTs downstream of the establishment of proper PCP.

Active Antioxidizing Particles for On-Demand Pressure-Driven Molecular Release.

Overproduced reactive oxygen species (ROS) are closely related to various health problems including inflammation, infection, and cancer. Abnormally high ROS levels can cause serious oxidative damage to biomolecules, cells, and tissues. A series of nano- or microsized particles has been developed to reduce the oxidative stress level by delivering antioxidant drugs. However, most systems are often plagued by slow molecular discharge, driven by diffusion. Herein, this study demonstrates the polymeric particles whose internal pressure can increase upon exposure to H2O2, one of the ROS, and in turn, discharge antioxidants actively. The on-demand pressurized particles are assembled by simultaneously encapsulating water-dispersible manganese oxide (MnO2) nanosheets and green tea derived epigallocatechin gallate (EGCG) molecules into a poly(lactic-co-glycolic acid) (PLGA) spherical shell. In the presence of H2O2, the MnO2 nanosheets in the PLGA particle generate oxygen gas by decomposing H2O2 and increase the internal pressure. The pressurized PLGA particles release antioxidative EGCG actively and, in turn, protect vascular and brain tissues from oxidative damage more effectively than the particles without MnO2 nanosheets. This H2O2 responsive, self-pressurizing particle system would be useful to deliver a wide array of molecular cargos in response to the oxidation level.

Melatonin Signal Transduction Pathways Require E-Box-Mediated Transcription of Per1 and Per2 to Reset the SCN Clock at Dusk.

Melatonin is released from the pineal gland into the circulatory system at night in the absence of light, acting as "hormone of darkness" to the brain and body. Melatonin also can regulate circadian phasing of the suprachiasmatic nucleus (SCN). During the day-to-night transition, melatonin exposure advances intrinsic SCN neural activity rhythms via the melatonin type-2 (MT2) receptor and downstream activation of protein kinase C (PKC). The effects of melatonin on SCN phasing have not been linked to daily changes in the expression of core genes that constitute the molecular framework of the circadian clock. Using real-time RT-PCR, we found that melatonin induces an increase in the expression of two clock genes, Period 1 (Per1) and Period 2 (Per2). This effect occurs at CT 10, when melatonin advances SCN phase, but not at CT 6, when it does not. Using anti-sense oligodeoxynucleotides (α ODNs) to Per 1 and Per 2, as well as to E-box enhancer sequences in the promoters of these genes, we show that their specific induction is necessary for the phase-altering effects of melatonin on SCN neural activity rhythms in the rat. These effects of melatonin on Per1 and Per2 were mediated by PKC. This is unlike day-active non-photic signals that reset the SCN clock by non-PCK signal transduction mechanisms and by decreasing Per1 expression. Rather, this finding extends roles for Per1 and Per2, which are critical to photic phase-resetting, to a nonphotic zeitgeber, melatonin, and suggest that the regulation of these clock gene transcripts is required for clock resetting by diverse regulatory cues.

Cell biology. Reversible centriole depletion with an inhibitor of Polo-like kinase 4.

Centrioles are ancient organelles that build centrosomes, the major microtubule-organizing centers of animal cells. Extra centrosomes are a common feature of cancer cells. To investigate the importance of centrosomes in the proliferation of normal and cancer cells, we developed centrinone, a reversible inhibitor of Polo-like kinase 4 (Plk4), a serine-threonine protein kinase that initiates centriole assembly. Centrinone treatment caused centrosome depletion in human and other vertebrate cells. Centrosome loss irreversibly arrested normal cells in a senescence-like G1 state by a p53-dependent mechanism that was independent of DNA damage, stress, Hippo signaling, extended mitotic duration, or segregation errors. In contrast, cancer cell lines with normal or amplified centrosome numbers could proliferate indefinitely after centrosome loss. Upon centrinone washout, each cancer cell line returned to an intrinsic centrosome number "set point." Thus, cells with cancer-associated mutations fundamentally differ from normal cells in their response to centrosome loss.

Radial intercalation is regulated by the Par complex and the microtubule-stabilizing protein CLAMP/Spef1.

The directed movement of cells is critical for numerous developmental and disease processes. A developmentally reiterated form of migration is radial intercalation; the process by which cells move in a direction orthogonal to the plane of the tissue from an inner layer to an outer layer. We use the radial intercalation of cells into the skin of Xenopus laevis embryos as a model to study directed cell migration within an epithelial tissue. We identify a novel function for both the microtubule-binding protein CLAMP and members of the microtubule-regulating Par complex during intercalation. Specifically, we show that Par3 and aPKC promote the apical positioning of centrioles, whereas CLAMP stabilizes microtubules along the axis of migration. We propose a model in which the Par complex defines the orientation of apical migration during intercalation and in which subcellular localization of CLAMP promotes the establishment of an axis of microtubule stability required for the active migration of cells into the outer epithelium.

Comparing label-free quantitative peptidomics approaches to characterize diurnal variation of peptides in the rat suprachiasmatic nucleus.

Mammalian circadian rhythm is maintained by the suprachiasmatic nucleus (SCN) via an intricate set of neuropeptides and other signaling molecules. In this work, peptidomic analyses from two times of day were examined to characterize variation in SCN peptides using three different label-free quantitation approaches: spectral count, spectra index and SIEVE. Of the 448 identified peptides, 207 peptides were analyzed by two label-free methods, spectral count and spectral index. There were 24 peptides with significant (adjusted p-value < 0.01) differential peptide abundances between daytime and nighttime, including multiple peptides derived from secretogranin II, cocaine and amphetamine regulated transcript, and proprotein convertase subtilisin/kexin type 1 inhibitor. Interestingly, more peptides were analyzable and had significantly different abundances between the two time points using the spectral count and spectral index methods than with a prior analysis using the SIEVE method with the same data. The results of this study reveal the importance of using the appropriate data analysis approaches for label-free relative quantitation of peptides. The detection of significant changes in so rich a set of neuropeptides reflects the dynamic nature of the SCN and the number of influences such as feeding behavior on circadian rhythm. Using spectral count and spectral index, peptide level changes are correlated to time of day, suggesting their key role in circadian function.

Deuterosome-mediated centriole biogenesis.

The ability of cells to faithfully duplicate their two centrioles once per cell cycle is critical for proper mitotic progression and chromosome segregation. Multiciliated cells represent an interesting variation of centriole duplication in that these cells generate greater than 100 centrioles, which form the basal bodies of their motile cilia. This centriole amplification is proposed to require a structure termed the deuterosome, thought to be capable of promoting de novo centriole biogenesis. Here, we begin to molecularly characterize the deuterosome and identify it as a site for the localization of Cep152, Plk4, and SAS6. Additionally we identify CCDC78 as a centriole-associated and deuterosome protein that is essential for centriole amplification. Overexpression of Cep152, but not Plk4, SAS6, or CCDC78, drives overamplification of centrioles. However, in CCDC78 morphants, Cep152 fails to localize to the deuterosome and centriole biogenesis is impaired, indicating that CCDC78-mediated recruitment of Cep152 is required for deuterosome-mediated centriole biogenesis.

Quantitative peptidomics for discovery of circadian-related peptides from the rat suprachiasmatic nucleus.

In mammals the suprachiasmatic nucleus (SCN), the master circadian clock, is sensitive to light input via the optic chiasm and synchronizes many daily biological rhythms. Here we explore variations in the expression levels of neuropeptides present in the SCN of rats using a label-free quantification approach that is based on integrating peak intensities between daytime, Zeitgeber time (ZT) 6, and nighttime, ZT 18. From nine analyses comparing the levels between these two time points, 10 endogenous peptides derived from eight prohormones exhibited significant differences in their expression levels (adjusted p-value <0.05). Of these, seven peptides derived from six prohormones, including GRP, PACAP, and CART, exhibited ≥ 30% increases at ZT 18, and the VGRPEWWMDYQ peptide derived from proenkephalin A showed a >50% increase at nighttime. Several endogenous peptides showing statistically significant changes in this study have not been previously reported to alter their levels as a function of time of day, nor have they been implicated in prior functional SCN studies. This information on peptide expression changes serves as a resource for discovering unknown peptide regulators that affect circadian rhythms in the SCN.

A hyphenated optical trap capillary electrophoresis laser induced native fluorescence system for single-cell chemical analysis.

Single-cell measurements allow a unique glimpse into cell-to-cell heterogeneity; even small changes in selected cells can have a profound impact on an organism's physiology. Here an integrated approach to single-cell chemical sampling and assay are described. Capillary electrophoresis (CE) with laser-induced native fluorescence (LINF) has the sensitivity to characterize natively fluorescent indoles and catechols within individual cells. While the separation and detection approaches are well established, the sampling and injection of individually selected cells requires new approaches. We describe an optimized system that interfaces a single-beam optical trap with CE and multichannel LINF detection. A cell is localized within the trap and then the capillary inlet is positioned near the cell using a computer-controlled micromanipulator. Hydrodynamic injection allows cell lysis to occur within the capillary inlet, followed by the CE separation and LINF detection. The use of multiple emission wavelengths allows improved analyte identification based on differences in analyte fluorescence emission profiles and migration time. The system enables injections of individual rat pinealocytes and quantification of their endogenous indoles, including serotonin, N-acetyl-serotonin, 5-hydroxyindole-3-acetic acid, tryptophol and others. The amounts detected in individual cells incubated in 5-hydroxytryptophan ranged from 10(-14) mol to 10(-16) mol, an order of magnitude higher than observed in untreated pinealocytes.

Heparin cofactor II: discovery, properties, and role in controlling vascular homeostasis.

Heparin cofactor II (HCII) is a serine protease inhibitor (serpin) found in high concentrations in human plasma. Despite its discovery >30 years ago, its physiological function is still poorly understood. It is known to inhibit thrombin, the predominant coagulation protease, and HCII-thrombin complexes have been found in plasma, yet it is thought to contribute little to normal hemostasis. However, thrombin has several other physiological functions, and therefore many biological roles for HCII need consideration. The unique structure and mechanism of action of HCII have helped guide our understanding of HCII. In particular, HCII binds many glycosaminoglycans (GAGs) such as heparin and heparin sulfate as well as several different polyanions to enhance its inhibition of thrombin. Distinctly, HCII is able to use the GAG dermatan sulfate for accelerated thrombin inhibition. Dermatan sulfate is found in high concentrations in the walls of blood vessels as well as in placental tissue. This knowledge has led to research indicating that HCII may play a protective role in atherosclerosis and placental thrombosis. Additionally, pharmaceuticals are being developed that use the dermatan sulfate activation of HCII for anticoagulation. Although much research is still needed to fully understand HCII, this humble protein may have significant impact in our medical future. This article reviews the laboratory history, protein characteristics, structure-activity relationships, protease inhibition, physiological function, and medical relevance of HCII in hopes of regenerating interest in this sometimes forgotten serpin.

Direct cellular peptidomics of hypothalamic neurons.

The chemical complexity of cell-to-cell communication has emerged as a fundamental challenge to understanding brain systems. This is certainly true for the hypothalamus, where neuropeptide signals are heterogeneous, localized and dynamic. Thus far, most hypothalamic peptidomic studies have centered on the entire structure; however, recent advances in collection strategies and analytical technologies have enabled direct, high-resolution peptidomic profiles focused on two regions of interest, the suprachiasmatic and supraoptic nuclei, including their sub-regions and individual cells. Suites of peptides now can be identified and probed for function. High spatial and analytical sensitivities reveal that discrete hypothalamic nuclei have distinct peptidomic signatures. Peptidomic discovery not only reveals unanticipated complexity, but also peptides previously unknown that act as key circuit components. Analysis of tissue releasates identifies peptides secreted into the extracellular environment and available for transmitting intercellular signals. Direct sampling techniques define peptide-releasate profiles in spatial, temporal and event-dependent patterns. These approaches are providing remarkable new insights into the complexity of neuropeptidergic cell-to-cell signaling central to neuroendocrine physiology.

Circadian integration of glutamatergic signals by little SAAS in novel suprachiasmatic circuits.

BACKGROUND: Neuropeptides are critical integrative elements within the central circadian clock in the suprachiasmatic nucleus (SCN), where they mediate both cell-to-cell synchronization and phase adjustments that cause light entrainment. Forward peptidomics identified little SAAS, derived from the proSAAS prohormone, among novel SCN peptides, but its role in the SCN is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Little SAAS localization and co-expression with established SCN neuropeptides were evaluated by immunohistochemistry using highly specific antisera and stereological analysis. Functional context was assessed relative to c-FOS induction in light-stimulated animals and on neuronal circadian rhythms in glutamate-stimulated brain slices. We found that little SAAS-expressing neurons comprise the third most abundant neuropeptidergic class (16.4%) with unusual functional circuit contexts. Little SAAS is localized within the densely retinorecipient central SCN of both rat and mouse, but not the retinohypothalamic tract (RHT). Some little SAAS colocalizes with vasoactive intestinal polypeptide (VIP) or gastrin-releasing peptide (GRP), known mediators of light signals, but not arginine vasopressin (AVP). Nearly 50% of little SAAS neurons express c-FOS in response to light exposure in early night. Blockade of signals that relay light information, via NMDA receptors or VIP- and GRP-cognate receptors, has no effect on phase delays of circadian rhythms induced by little SAAS. CONCLUSIONS/SIGNIFICANCE: Little SAAS relays signals downstream of light/glutamatergic signaling from eye to SCN, and independent of VIP and GRP action. These findings suggest that little SAAS forms a third SCN neuropeptidergic system, processing light information and activating phase-shifts within novel circuits of the central circadian clock.

Temporally restricted role of retinal PACAP: integration of the phase-advancing light signal to the SCN.

Circadian rhythms in physiology and behavior are temporally synchronized to the day/night cycle through the action of light on the circadian clock. In mammals, transduction of the photic signal reaching the circadian oscillator in the suprachiasmatic nucleus (SCN) occurs through the release of glutamate and pituitary adenylate cyclase-activating peptide (PACAP). The authors' study aimed at clarifying the role played by PACAP in photic resetting and entrainment. They investigated the circadian response to light of PACAPnullmice lacking the 5th exon of the PACAP coding sequence. Specifically, they examined free-running rhythms, entrainment to 12-h light:12-h dark (LD)cycles, the phase-response curve (PRC) to single light pulses, entrainment to a23-h T-cycle, re-entrainment to 6-h phase shifts in LD cycles, and light-induced c-Fos expression. PACAP-null and wild-type mice show similar free-running periods and similar entrainment to 12:12 LD cycles. However, the PRC of PACAP-null mice lacks a phase-advance portion. Surprisingly, despite the absence of phase advance to single light pulses, PACAP-null mice are able to entrain to a 23-h T-cycle, but with a significantly longer phase angle of entrainment than wild types. In addition, PACAP-null mice re-entrain more slowly to a 6-h phase advance of the LD cycle. Nevertheless, induction of c-Fos by light in late night is normal. In all experiments, PACAP-null mice show specific behavioral impairments in response to phase-advancing photic stimuli. These results suggest that PACAP is required for the normal integration of the phase advancing light signal by the SCN.