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Daily changes in ambient illumination act as important time of day cues which are pivotal for aligning internal circadian clocks to external time. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), generally considered specialized for encoding light intensity (irradiance), are critical to this photoentrainment process. However, ipRGCs also convey information from conventional photoreceptor cells, the rods and cones. Here we review data from animal studies identifying the nature and roles of rod and cone signaling to the suprachiasmatic nucleus (SCN) circadian clock including evidence that visual features other than irradiance (color, spatiotemporal variations in light intensity) may influence photoentrainment or other SCN-dependent functions. Finally we consider the extent to which these findings from animal studies might similarly apply to human circadian function., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Mammalian circadian rhythms are orchestrated by a master pacemaker in the hypothalamic suprachiasmatic nuclei (SCN), which receives information about the 24 h light-dark cycle from the retina. The accepted function of this light signal is to reset circadian phase in order to ensure appropriate synchronization with the celestial day. Here, we ask whether light also impacts another key property of the circadian oscillation, its amplitude. To this end, we measured circadian rhythms in behavioral activity, body temperature, and SCN electrophysiological activity in the diurnal murid rodent Rhabdomys pumilio following stable entrainment to 12:12 light-dark cycles at four different daytime intensities (ranging from 18 to 1,900 lx melanopic equivalent daylight illuminance). R. pumilio showed strongly diurnal activity and body temperature rhythms in all conditions, but measures of rhythm robustness were positively correlated with daytime irradiance under both entrainment and subsequent free run. Whole-cell and extracellular recordings of electrophysiological activity in ex vivo SCN revealed substantial differences in electrophysiological activity between dim and bright light conditions. At lower daytime irradiance, daytime peaks in SCN spontaneous firing rate and membrane depolarization were substantially depressed, leading to an overall marked reduction in the amplitude of circadian rhythms in spontaneous activity. Our data reveal a previously unappreciated impact of daytime light intensity on SCN physiology and the amplitude of circadian rhythms and highlight the potential importance of daytime light exposure for circadian health., Competing Interests: Competing interest statement: T.M.B. and R.J.L. have received investigator-initiated grant funding from Signify (formerly Philips Lighting). R.J.L. has received honoraria from Samsung Electronics., (Copyright © 2021 the Author(s). Published by PNAS.)

Intrinsically photosensitive retinal ganglion cells convey intrinsic, melanopsin-based, photoreceptive signals alongside those produced by rods and cones to the suprachiasmatic nucleus (SCN) circadian clock. To date, experimental data suggest that melanopsin plays a more significant role in measuring ambient light intensity than cone photoreception. Such studies have overwhelmingly used diffuse light stimuli, whereas light intensity in the world around us varies across space and time. Here, we investigated the extent to which melanopsin or cone signals support circadian irradiance measurements in the presence of naturalistic spatiotemporal variations in light intensity. To address this, we first presented high- and low-contrast movies to anaesthetised mice whilst recording extracellular electrophysiological activity from the SCN. Using a mouse line with altered cone sensitivity (Opn1mw R mice) and multispectral light sources we then selectively varied irradiance of the movies for specific photoreceptor classes. We found that steps in melanopic irradiance largely account for the light induced-changes in SCN activity over a range of starting light intensities and in the presence of spatiotemporal modulation. By contrast, cone-directed changes in irradiance only influenced SCN activity when spatiotemporal contrast was low. Consistent with these findings, under housing conditions where we could independently adjust irradiance for melanopsin versus cones, the period lengthening effects of constant light on circadian rhythms in behaviour were reliably determined by melanopic irradiance, regardless of irradiance for cones. These data add to the growing evidence that modulating effective irradiance for melanopsin is an effective strategy for controlling the circadian impact of light., (© 2021 The Authors. Journal of Pineal Research published by John Wiley & Sons Ltd.)

Electrical activity in the heart exhibits 24-hour rhythmicity, and potentially fatal arrhythmias are more likely to occur at specific times of day. Here, we demonstrate that circadian clocks within the brain and heart set daily rhythms in sinoatrial (SA) and atrioventricular (AV) node activity, and impose a time-of-day dependent susceptibility to ventricular arrhythmia. Critically, the balance of circadian inputs from the autonomic nervous system and cardiomyocyte clock to the SA and AV nodes differ, and this renders the cardiac conduction system sensitive to decoupling during abrupt shifts in behavioural routine and sleep-wake timing. Our findings reveal a functional segregation of circadian control across the heart's conduction system and inherent susceptibility to arrhythmia.

Background: Daily variations in mammalian physiology are under control of a central clock in the suprachiasmatic nucleus (SCN). SCN timing signals are essential for coordinating cellular clocks and associated circadian variations in cell and tissue function across the body; however, direct SCN projections primarily target a restricted set of hypothalamic and thalamic nuclei involved in physiological and behavioural control. The role of the SCN in driving rhythmic activity in these targets remains largely unclear. Here, we address this issue via multielectrode recording and manipulations of SCN output in adult mouse brain slices., Results: Electrical stimulation identifies cells across the midline hypothalamus and ventral thalamus that receive inhibitory input from the SCN and/or excitatory input from the retina. Optogenetic manipulations confirm that SCN outputs arise from both VIP and, more frequently, non-VIP expressing cells and that both SCN and retinal projections almost exclusively target GABAergic downstream neurons. The majority of midline hypothalamic and ventral thalamic neurons exhibit circadian variation in firing and those receiving inhibitory SCN projections consistently exhibit peak activity during epochs when SCN output is low. Physical removal of the SCN confirms that neuronal rhythms in ~ 20% of the recorded neurons rely on central clock input but also reveals many neurons that can express circadian variation in firing independent of any SCN input., Conclusions: We identify cell populations across the midline hypothalamus and ventral thalamus exhibiting SCN-dependent and independent rhythms in neural activity, providing new insight into the mechanisms by which the circadian system generates daily physiological rhythms.

Ocular light drives a range of nonvisual responses in humans including suppression of melatonin secretion and circadian phase resetting. These responses are driven by intrinsically photosensitive retinal ganglion cells (ipRGCs) which combine intrinsic, melanopsin-based, phototransduction with extrinsic rod/cone-mediated signals. As a result of this arrangement, it has remained unclear how best to quantify light to predict its nonvisual effects. To address this, we analysed data from nineteen different laboratory studies that measured melatonin suppression, circadian phase resetting and/or alerting responses in humans to a wide array of stimulus types, intensities and durations with or without pupil dilation. Using newly established SI-compliant metrics to quantify ipRGC-influenced responses to light, we show that melanopic illuminance consistently provides the best available predictor for responses of the human circadian system. In almost all cases, melanopic illuminance is able to fully account for differences in sensitivity to stimuli of varying spectral composition, acting to drive responses that track variations in illumination characteristic of those encountered over civil twilight (~1-1000 lux melanopic equivalent daylight illuminance). Collectively, our data demonstrate widespread utility of melanopic illuminance as a metric for predicting the circadian impact of environmental illumination. These data therefore provide strong support for the use of melanopic illuminance as the basis for guidelines that seek to regulate light exposure to benefit human health and to inform future lighting design., (© 2020 The Authors. Journal of Pineal Research published by John Wiley & Sons Ltd.)

The suprachiasmatic nucleus (SCN) circadian clock is critical for optimising daily cycles in mammalian physiology and behaviour. The roles of the various SCN cell types in communicating timing information to downstream physiological systems remain incompletely understood, however. In particular, while vasoactive intestinal polypeptide (VIP) signalling is essential for SCN function and whole animal circadian rhythmicity, the specific contributions of VIP cell output to physiological control remains uncertain. Here we reveal a key role for SCN VIP cells in central clock output. Using multielectrode recording and optogenetic manipulations, we show that VIP neurons provide coordinated daily waves of GABAergic input to target cells across the paraventricular hypothalamus and ventral thalamus, supressing their activity during the mid to late day. Using chemogenetic manipulation, we further demonstrate specific roles for this circuitry in the daily control of heart rate and corticosterone secretion, collectively establishing SCN VIP cells as influential regulators of physiological timing.

In humans, short-wavelength light evokes larger circadian responses than longer wavelengths [1-3]. This reflects the fact that melanopsin, a key contributor to circadian assessments of light intensity, most efficiently captures photons around 480 nm [4-8] and gives rise to the popular view that "blue" light exerts the strongest effects on the clock. However, in the natural world, there is often no direct correlation between perceived color (as reported by the cone-based visual system) and melanopsin excitation. Accordingly, although the mammalian clock does receive cone-based chromatic signals [9], the influence of color on circadian responses to light remains unclear. Here, we define the nature and functional significance of chromatic influences on the mouse circadian system. Using polychromatic lighting and mice with altered cone spectral sensitivity (Opn1mw R ), we generate conditions that differ in color (i.e., ratio of L- to S-cone opsin activation) while providing identical melanopsin and rod activation. When biased toward S-opsin activation (appearing "blue"), these stimuli reliably produce weaker circadian behavioral responses than those favoring L-opsin ("yellow"). This influence of color (which is absent in animals lacking cone phototransduction; Cnga3 -/- ) aligns with natural changes in spectral composition over twilight, where decreasing solar angle is accompanied by a strong blue shift [9-11]. Accordingly, we find that naturalistic color changes support circadian alignment when environmental conditions render diurnal variations in light intensity weak/ambiguous sources of timing information. Our data thus establish how color contributes to circadian entrainment in mammals and provide important new insight to inform the design of lighting environments that benefit health., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

During dusk and dawn, the ambient illumination undergoes drastic changes in irradiance (or intensity) and spectrum (or color). While the former is a well-studied factor in synchronizing behavior and physiology to the earth's 24-h rotation, color sensitivity in the regulation of circadian rhythms has not been systematically studied. Drawing on the concept of color opponency, a well-known property of image-forming vision in many vertebrates (including humans), we consider how the spectral shifts during twilight are encoded by a color-opponent sensory system for non-image-forming (NIF) visual functions, including phase shifting and melatonin suppression. We review electrophysiological evidence for color sensitivity in the pineal/parietal organs of fish, amphibians and reptiles, color coding in neurons in the circadian pacemaker in mice as well as sporadic evidence for color sensitivity in NIF visual functions in birds and mammals. Together, these studies suggest that color opponency may be an important modulator of light-driven physiological and behavioral responses., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A direct retinal projection targets the suprachiasmatic nucleus (SCN) (an important hypothalamic control center). The accepted function of this projection is to convey information about ambient light (irradiance) to synchronize the SCN's endogenous circadian clock with local time and drive the diurnal variations in physiology and behavior [1-4]. Here, we report that it also renders the SCN responsive to visual images. We map spatial receptive fields (RFs) for SCN neurons and find that only a minority are excited (or inhibited) by light from across the scene as expected for irradiance detectors. The most commonly encountered units have RFs with small excitatory centers, combined with very extensive inhibitory surrounds that reduce their sensitivity to global changes in light in favor of responses to spatial patterns. Other units have larger excitatory RF centers, but these always cover a coherent region of visual space, implying visuotopic order at the single-unit level. Approximately 75% of light-responsive SCN units modulate their firing according to simple spatial patterns (drifting or inverting gratings) without changes in irradiance. The time-averaged firing rate of the SCN is modestly increased under these conditions, but including spatial contrast did not significantly alter the circadian phase resetting efficiency of light. Our data indicate that the SCN contains information about irradiance and spatial patterns. This newly appreciated sensory capacity provides a mechanism by which behavioral and physiological systems downstream of the SCN could respond to visual images [5]., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The epithalamic lateral habenula (LHb) is implicated as part of the mammalian brain's circadian system. Anatomical evidence suggests that the LHb receives extrinsic circadian timing cues from retinal ganglion cells and the master clock in the suprachiasmatic nuclei (SCN). Intriguingly, some LHb neurones contain the molecular circadian clock, but it is unclear if and how intrinsic and extrinsic circadian processes influence neuronal activity in the mouse LHb. Here, using an in vitro brain slice preparation isolating the LHb from the SCN, we show through whole-cell patch-clamp recordings that LHb neurones exhibit heterogeneity in their resting state, but the majority spontaneously fire action potentials (APs). Discharge rate of APs varied from low firing in the early day to higher firing later in the day and was absent in LHb brain slices prepared from Cry1(-/-)Cry2(-/-) mice that lack a functional molecular clock. Low amplitude circadian oscillations in the molecular circadian clock were also monitored in LHb brain slices, but were absent in Cry1(-/-)Cry2(-/-) LHb brain tissue. A putative neurochemical output signal of the SCN, prokineticin 2 (PK2), inhibited some LHb neurones by elevating the frequency of GABA release in the LHb. Using multi-electrode recordings in vivo, we found that LHb neurones sluggishly respond to retinal illumination, suggesting that they receive such information through polysynaptic processes. In summary, our results show for the first time that intrinsic circadian signals are important for regulating LHb neuronal state, while the SCN-derived signal PK2 is less influential. Moreover, we demonstrate that mouse LHb neurones have access to and can respond to visual input, but such signals are unlikely to be directly communicated to the LHb. Broadly, these findings raise the possibility that intrinsic circadian signals are likely to be influential in shaping LHb contributions to cognition and emotionality., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

Within the core molecular clock, protein phosphorylation and degradation play a vital role in determining circadian period. The 'after-hours' (Afh) mutation in mouse slows the degradation of the core clock protein Cryptochrome, lengthening the period of the molecular clock in the suprachiasmatic nuclei (SCN) and behavioural wheel-running rhythms. However, we do not yet know how the Afh mutation affects other aspects of physiology or the activity of circadian oscillators in other brain regions. Here we report that daily rhythms of metabolism and ingestive behaviours are altered in these animals, as are PERIOD2::LUCIFERASE (PER2::LUC) rhythms in mediobasal hypothalamic nuclei, which influence these behaviours. Overall there is a trend towards period lengthening and a decrease in amplitude of PER2::LUC rhythms throughout the brain. Imaging of single cells from the arcuate and dorsomedial hypothalamic nuclei revealed this reduction in tissue oscillator amplitude to be due to a decrease in the amplitude, rather than a desynchrony, of single cells. Consistent with existing models of oscillator function, this cellular phenotype was associated with a greater susceptibility to phase-shifting stimuli in vivo and in vitro, with light evoking high-amplitude Type 0 resetting in Afh mutant mice. Together, these findings reveal unexpected consequences of the Afh mutation on the amplitude and synchrony of individual cellular oscillators in the SCN.

Environmental illumination profoundly influences mammalian physiology and behaviour through actions on a master circadian oscillator in the suprachiasmatic nuclei (SCN) and other hypothalamic nuclei. The retinal and central mechanisms that shape daily patterns of light-evoked and spontaneous activity in this network of hypothalamic cells are still largely unclear. Similarly, the exact nature of the sensory information conveyed by such cells is unresolved. Here we set out to address these issues, through multielectrode recordings from the hypothalamus of red cone knockin mice (Opn1mwR). With this powerful mouse model, the photoreceptive origins of any response can be readily identified on the basis of their relative sensitivity to short and long wavelength light. Our experiments revealed that the firing pattern of many hypothalamic cells was influenced by changes in light levels and/or according to the steady state level of illumination. These ‘contrast' and ‘irradiance' responses were driven primarily by cone and melanopsin photoreceptors respectively, with rods exhibiting a much more subtle influence. Individual hypothalamic neurons differentially sampled from these information streams, giving rise to four distinct response types. The most common response phenotype in the SCN itself was sustained activation. Cells with this behaviour responded to all three photoreceptor classes in a manner consistent with their distinct contributions to circadian photoentrainment. These ‘sustained' cells were also unique in our sample in expressing circadian firing patterns with highest activity during the mid projected day. Surprisingly, we also found a minority of SCN neurons that lacked the melanopsin-derived irradiance signal and responded only to light transitions, allowing for the possibility that rod–cone contrast signals may be routed to SCN output targets without influencing neighbouring circadian oscillators. Finally, an array of cells extending throughout the periventricular hypothalamus and ventral thalamus were excited or inhibited solely according to the activity of melanopsin. These cells appeared to convey a filtered version of the visual signal, suitable for modulating physiology/behaviour purely according to environmental irradiance. In summary, these findings reveal unexpectedly widespread hypothalamic cell populations encoding distinct qualities of visual information.

Targeted deletion of IA-2 and IA-2beta, major autoantigens in type 1 diabetes and transmembrane secretory vesicle proteins, results in impaired secretion of hormones and neurotransmitters. In the present study, we evaluated the effect of these deletions on daily rhythms in blood pressure, heart rate, core body temperature, and spontaneous physical and neuronal activity. We found that deletion of both IA-2 and IA-2beta profoundly disrupts the usual diurnal variation of each of these parameters, whereas the deletion of either IA-2 or IA-2beta alone did not produce a major change. In situ hybridization revealed that IA-2 and IA-2beta transcripts are highly but nonrhythmically expressed in the suprachiasmatic nuclei, the site of the brain's master circadian oscillator. Electrophysiological studies on tissue slices from the suprachiasmatic nuclei showed that disruption of both IA-2 and IA-2beta results in significant alterations in neuronal firing. From these studies, we concluded that deletion of IA-2 and IA-2beta, structural proteins of secretory vesicles and modulators of neuroendocrine secretion, has a profound effect on the circadian system.

Background: In mammals, the synchronized activity of cell autonomous clocks in the suprachiasmatic nuclei (SCN) enables this structure to function as the master circadian clock, coordinating daily rhythms in physiology and behavior. However, the dominance of this clock has been challenged by the observations that metabolic duress can over-ride SCN controlled rhythms, and that clock genes are expressed in many brain areas, including those implicated in the regulation of appetite and feeding. The recent development of mice in which clock gene/protein activity is reported by bioluminescent constructs (luciferase or luc) now enables us to track molecular oscillations in numerous tissues ex vivo. Consequently we determined both clock activities and responsiveness to metabolic perturbations of cells and tissues within the mediobasal hypothalamus (MBH), a site pivotal for optimal internal homeostatic regulation., Results: Here we demonstrate endogenous circadian rhythms of PER2::LUC expression in discrete subdivisions of the arcuate (Arc) and dorsomedial nuclei (DMH). Rhythms resolved to single cells did not maintain long-term synchrony with one-another, leading to a damping of oscillations at both cell and tissue levels. Complementary electrophysiology recordings revealed rhythms in neuronal activity in the Arc and DMH. Further, PER2::LUC rhythms were detected in the ependymal layer of the third ventricle and in the median eminence/pars tuberalis (ME/PT). A high-fat diet had no effect on the molecular oscillations in the MBH, whereas food deprivation resulted in an altered phase in the ME/PT., Conclusion: Our results provide the first single cell resolution of endogenous circadian rhythms in clock gene expression in any intact tissue outside the SCN, reveal the cellular basis for tissue level damping in extra-SCN oscillators and demonstrate that an oscillator in the ME/PT is responsive to changes in metabolism.

The coordinated activity of thousands of cellular oscillators in the suprachiasmatic nuclei (SCN) temporally regulates mammalian physiology to anticipate daily environmental changes across the seasons. The phasing of clock gene expression varies according to anatomical location in the SCN and is thought to encode photoperiodic information. However, it is unclear whether similar variations in phase occur in the electrical activity of SCN neurons, a measure of both intraSCN signaling and clock output. To address this, we recorded single-unit and multiunit activity (SUA/MUA) from dorsal and ventral subregions of the middle level of the rostrocaudal axis of the SCN in coronal brain slices prepared from mice housed under different photoperiods. We demonstrate that under a symmetrical (12 h light:12 h dark) photoperiod, cells in the dorsal SCN are less tightly synchronized than those in the ventral SCN. Comparison of recordings made from mice under short (8 h light:16 h dark) or long (16 h light:8 h dark) photoperiods shows that the phase distribution of ventral, but not dorsal, SCN neurons expands with increasing day length. Conversely, the duration that individual neurons are active increases in dorsal, but not ventral, SCN under long days. These data indicate that in the ventral SCN photoperiod is encoded at the network level, while this coding occurs at the level of individual cells in the dorsal SCN.

The intrinsic period of circadian clocks is their defining adaptive property. To identify the biochemical mechanisms whereby casein kinase1 (CK1) determines circadian period in mammals, we created mouse null and tau mutants of Ck1 epsilon. Circadian period lengthened in CK1epsilon-/-, whereas CK1epsilon(tau/tau) shortened circadian period of behavior in vivo and suprachiasmatic nucleus firing rates in vitro, by accelerating PERIOD-dependent molecular feedback loops. CK1epsilon(tau/tau) also accelerated molecular oscillations in peripheral tissues, revealing its global role in circadian pacemaking. CK1epsilon(tau) acted by promoting degradation of both nuclear and cytoplasmic PERIOD, but not CRYPTOCHROME, proteins. Together, these whole-animal and biochemical studies explain how tau, as a gain-of-function mutation, acts at a specific circadian phase to promote degradation of PERIOD proteins and thereby accelerate the mammalian clockwork in brain and periphery.

The circadian pacemaker in the suprachiasmatic nuclei (SCN) controls endogenous near 24-h physiological and behavioral rhythms in metabolism, neuroendocrine function, and locomotor activity. Recently, we showed that vasoactive intestinal polypeptide (VIP) and its receptor, VPAC(2) are critical to the intercellular communication between individual SCN neurons, and appropriate synchronization and phasing of these oscillatory cells. Mice defective in VIP signaling manifest grossly impaired circadian rhythms of SCN neuronal firing activity and are typically unable to maintain rhythmic wheel-running behavior in the absence of external time cues. Here we report that daily rhythms of metabolism and feeding behavior are also overtly altered in these animals. Under diurnal conditions (12:12-h light-dark; LD), metabolic and feeding rhythms are advanced in mice lacking either VIP or VPAC(2) receptor expression, peaking in the late day, rather than early night, as observed in wild-type mice. When placed in constant light (LL), both VIP-deficient and VPAC(2) receptor-knockout mice exhibit dampening of metabolic and feeding rhythms, which deteriorate after a few days. In addition, overall metabolic rate is greatly reduced in VPAC(2)-knockout mice, when compared with wild-type mice, regardless of lighting condition. The advancement of metabolic and feeding rhythms in these mice under LD suggests that these rhythms are less sensitive to masking by light. These results demonstrate that altering SCN function not only affects neuronal and wheel-running activity rhythms but also dramatically impairs temporal regulation of metabolism and feeding.

In mammals, an internal timekeeping mechanism located in the suprachiasmatic nuclei (SCN) orchestrates a diverse array of neuroendocrine and physiological parameters to anticipate the cyclical environmental fluctuations that occur every solar day. Electrophysiological recording techniques have proved invaluable in shaping our understanding of how this endogenous clock becomes synchronized to salient environmental cues and appropriately coordinates the timing of a multitude of physiological rhythms in other areas of the brain and body. In this review we discuss the pioneering studies that have shaped our understanding of how this biological pacemaker functions, from input to output. Further, we highlight insights from new studies indicating that, more than just reflecting its oscillatory output, electrical activity within individual clock cells is a vital part of SCN clockwork itself.

Vasoactive intestinal polypeptide (VIP), acting via the VPAC(2) receptor, is a key signaling pathway in the suprachiasmatic nuclei (SCN), the master clock controlling daily rhythms in mammals. Most mice lacking functional VPAC(2) receptors are unable to sustain behavioral rhythms and lack detectable SCN electrical rhythms in vitro. Adult mice that do not produce VIP (VIP/PHI(-/-)) exhibit less severe alterations in wheel-running rhythms, but the effects of this deficiency on the amplitude, phasing, or periodicity of their SCN cellular rhythms are unknown. To investigate this, we used suction electrodes to extracellularly record multiple- and single-unit electrical activity in SCN brain slices from mice with varying degrees of VIP deficiency, ranging from wild-type (VIP/PHI(+/+)) to heterozygous (VIP/PHI(+/-)) and VIP/PHI(-/-) animals. We found decreasing proportions of rhythmic cells in SCN slices from VIP/PHI(+/+) ( approximately 91%, n = 23) through VIP/PHI(-/+) ( approximately 71%, n = 28) to VIP/PHI(-/-) mice (62%; n = 37) and a parallel trend toward decreasing amplitude in the remaining rhythmic cells. SCN neurons from VIP/PHI(-/-) mice exhibited a broad range in the period and phasing of electrical rhythms, concordant with the known alterations in their behavioral rhythms. Further, treatment of VIP/PHI(-/-) slices with a VPAC(2) receptor antagonist significantly reduced the proportion of oscillating neurons, suggesting that VPAC(2) receptors still become activated in the SCN of these mice. The results establish that VIP is important for appropriate periodicity and phasing of SCN neuronal rhythms and suggest that residual VPAC(2) receptor signaling promotes rhythmicity in adult VIP/PHI(-/-) mice.

The study of spontaneous neuronal discharge for several days in vitro poses substantial technical difficulties not readily addressed by existing methodologies. Here we describe a novel method for recording bioelectrical signals from brain slices, using media-filled, glass micropipettes stabilized at the recording interface by negative pressure. Such electrodes are durable, economical and easily constructed using standard laboratory equipment. Through these suction electrodes, we monitored approximately 24 h oscillations in spontaneous single and multiunit discharge from acutely prepared adult mouse suprachiasmatic nuclei (SCN) slices for up to 4 days in vitro, with very high success rates. Neuronal oscillations exhibited all the characteristics previously determined in SCN recordings using other techniques. Importantly, our electrode assembly enables SCN activity in acutely prepared brain slices to be monitored for substantially longer than with other methodologies. In summary, our data suggest that suction electrodes are likely to provide a favorable alternative to existing methods for recording long-term neuronal activity from brain slices.