Cell Signaling in the Circadian Pacemaker: New Insights from in vivo Imaging.

BACKGROUND: "One for all, and all for one," the famous rallying cry of the Three Musketeers, in Alexandre Dumas's popular novel, certainly applies to the 20,000 cells composing the suprachiasmatic nuclei (SCN). These cells work together to form the central clock that coordinates body rhythms in tune with the day-night cycle. Like virtually every body cell, individual SCN cells exhibit autonomous circadian oscillations, but this rhythmicity only reaches a high level of precision and robustness when the cells are coupled with their neighbors. Therefore, understanding the functional network organization of SCN cells beyond their core rhythmicity is an important issue in circadian biology. SUMMARY: The present review summarizes the main results from our recent study demonstrating the feasibility of recording SCN cells in freely moving mice and the significance of variations in intracellular calcium over several timescales. KEY MESSAGE: We discuss how in vivo imaging at the cell level will be pivotal to interrogate the mammalian master clock, in an integrated context that preserves the SCN network organization, with intact inputs and outputs.

Molecular and Cellular Networks in The Suprachiasmatic Nuclei.

Why do we experience the ailments of jetlag when we travel across time zones? Why is working night-shifts so detrimental to our health? In other words, why can't we readily choose and stick to non-24 h rhythms? Actually, our daily behavior and physiology do not simply result from the passive reaction of our organism to the external cycle of days and nights. Instead, an internal clock drives the variations in our bodily functions with a period close to 24 h, which is supposed to enhance fitness to regular and predictable changes of our natural environment. This so-called circadian clock relies on a molecular mechanism that generates rhythmicity in virtually all of our cells. However, the robustness of the circadian clock and its resilience to phase shifts emerge from the interaction between cell-autonomous oscillators within the suprachiasmatic nuclei (SCN) of the hypothalamus. Thus, managing jetlag and other circadian disorders will undoubtedly require extensive knowledge of the functional organization of SCN cell networks. Here, we review the molecular and cellular principles of circadian timekeeping, and their integration in the multi-cellular complexity of the SCN. We propose that new, in vivo imaging techniques now enable to address these questions directly in freely moving animals.

Complementary actions of dopamine D2 receptor agonist and anti-vegf therapy on tumoral vessel normalization in a transgenic mouse model.

Angiogenesis contributes in multiple ways to disease progression in tumors and reduces treatment efficiency. Molecular therapies targeting Vegf signaling combined with chemotherapy or other drugs exhibit promising results to improve efficacy of treatment. Dopamine has been recently proposed to be a novel safe anti-angiogenic drug that stabilizes abnormal blood vessels and increases therapeutic efficacy. Here, we aimed to identify a treatment to normalize tumoral vessels and restore normal blood perfusion in tumor tissue with a Vegf receptor inhibitor and/or a ligand of dopamine G protein-coupled receptor D2 (D2R). Dopamine, via its action on D2R, is an endogenous effector of the pituitary gland, and we took advantage of this system to address this question. We have used a previously described Hmga2/T mouse model developing haemorrhagic prolactin-secreting adenomas. In mutant mice, blood vessels are profoundly altered in tumors, and an aberrant arterial vascularization develops leading to the loss of dopamine supply. D2R agonist treatment blocks tumor growth, induces regression of the aberrant blood supply and normalizes blood vessels. A chronic treatment is able to restore the altered balance between pro- and anti-angiogenic factors. Remarkably, an acute treatment induces an upregulation of the stabilizing factor Angiopoietin 1. An anti-Vegf therapy is also effective to restrain tumor growth and improves vascular remodeling. Importantly, only the combination treatment suppresses intratumoral hemorrhage and restores blood vessel perfusion, suggesting that it might represent an attractive therapy targeting tumor vasculature. Similar strategies targeting other ligands of GPCRs involved in angiogenesis may identify novel therapeutic opportunities for cancer.

Plasticity of hypothalamic dopamine neurons during lactation results in dissociation of electrical activity and release.

Tuberoinfundibular dopamine (TIDA) neurons are the central regulators of prolactin (PRL) secretion. Their extensive functional plasticity allows a change from low PRL secretion in the non-pregnant state to the condition of hyperprolactinemia that characterizes lactation. To allow this rise in PRL, TIDA neurons are thought to become unresponsive to PRL at lactation and functionally silenced. Here we show that, contrary to expectations, the electrical properties of the system were not modified during lactation and that the neurons remained electrically responsive to a PRL stimulus, with PRL inducing an acute increase in their firing rate during lactation that was identical to that seen in non-pregnant mice. Furthermore, we show a long-term organization of TIDA neuron electrical activity with an harmonization of their firing rates, which remains intact during lactation. However, PRL-induced secretion of dopamine (DA) at the median eminence was strongly blunted during lactation, at least in part attributable to lack of phosphorylation of tyrosine hydroxylase, the key enzyme involved in DA synthesis. We therefore conclude that lactation, rather than involving electrical silencing of TIDA neurons, represents a condition of decoupling between electrical activity at the cell body and DA secretion at the median eminence.

Pituitary growth hormone network responses are sexually dimorphic and regulated by gonadal steroids in adulthood.

There are well-recognized sex differences in many pituitary endocrine axes, usually thought to be generated by gonadal steroid imprinting of the neuroendocrine hypothalamus. However, the recognition that growth hormone (GH) cells are arranged in functionally organized networks raises the possibility that the responses of the network are different in males and females. We studied this by directly monitoring the calcium responses to an identical GH-releasing hormone (GHRH) stimulus in populations of individual GH cells in slices taken from male and female murine GH-eGFP pituitary glands. We found that the GH cell network responses are sexually dimorphic, with a higher proportion of responding cells in males than in females, correlated with greater GH release from male slices. Repetitive waves of calcium spiking activity were triggered by GHRH in some males, but were never observed in females. This was not due to a permanent difference in the network architecture between male and female mice; rather, the sex difference in the proportions of GH cells responding to GHRH were switched by postpubertal gonadectomy and reversed with hormone replacements, suggesting that the network responses are dynamically regulated in adulthood by gonadal steroids. Thus, the pituitary gland contributes to the sexually dimorphic patterns of GH secretion that play an important role in differences in growth and metabolism between the sexes.

A Journey in the Brain's Clock: In Vivo Veritas?

The suprachiasmatic nuclei (SCN) of the hypothalamus contain the circadian pacemaker that coordinates mammalian rhythms in tune with the day-night cycle. Understanding the determinants of the intrinsic rhythmicity of this biological clock, its outputs, and resetting by environmental cues, has been a longstanding goal of the field. Integrated techniques of neurophysiology, including lesion studies and in vivo multi-unit electrophysiology, have been key to characterizing the rhythmic nature and outputs of the SCN in animal models. In parallel, reduced ex vivo and in vitro approaches have permitted us to unravel molecular, cellular, and multicellular mechanisms underlying the pacemaker properties of the SCN. New questions have emerged in recent years that will require combining investigation at a cell resolution within the physiological context of the living animal: What is the role of specific cell subpopulations in the SCN neural network? How do they integrate various external and internal inputs? What are the circuits involved in controlling other body rhythms? Here, we review what we have already learned about the SCN from in vivo studies, and how the recent development of new genetically encoded tools and cutting-edge imaging technology in neuroscience offers chronobiologists the opportunity to meet these challenges.

TSH Pulses Finely Tune Thyroid Hormone Release and TSH Receptor Transduction.

Detection of circulating TSH is a first-line test of thyroid dysfunction, a major health problem (affecting about 5% of the population) that, if untreated, can lead to a significant deterioration of quality of life and adverse effects on multiple organ systems. Human TSH levels display both pulsatile and (nonpulsatile) basal TSH secretion patterns; however, the importance of these in regulating thyroid function and their decoding by the thyroid is unknown. Here, we developed a novel ultra-sensitive ELISA that allows precise detection of TSH secretion patterns with minute resolution in mouse models of health and disease. We characterized the patterns of ultradian TSH pulses in healthy, freely behaving mice over the day-night cycle. Challenge of the thyroid axis with primary hypothyroidism because of iodine deficiency, a major cause of thyroid dysfunction worldwide, results in alterations of TSH pulsatility. Induction in mouse models of sequential TSH pulses that mimic ultradian TSH profiles in periods of minutes were more efficient than sustained rises in basal TSH levels at increasing both thyroid follicle cAMP levels, as monitored with a genetically encoded cAMP sensor, and circulating thyroid hormone. Hence, this mouse TSH assay provides a powerful tool to decipher how ultradian TSH pulses encode thyroid outcomes and to uncover hidden parameters in the TSH-thyroid hormone set-point in health and disease.

Nested calcium dynamics support daily cell unity and diversity in the suprachiasmatic nuclei of free-behaving mice.

The suprachiasmatic nuclei (SCN) of the anterior hypothalamus host the circadian pacemaker that synchronizes mammalian rhythms with the day-night cycle. SCN neurons are intrinsically rhythmic, thanks to a conserved cell-autonomous clock mechanism. In addition, circuit-level emergent properties confer a unique degree of precision and robustness to SCN neuronal rhythmicity. However, the multicellular functional organization of the SCN is not yet fully understood. Indeed, although SCN neurons are well-coordinated, experimental evidences indicate that some neurons oscillate out of phase in SCN explants, and possibly to a larger extent in vivo. Here, to tackle this issue we used microendoscopic Ca2+ i imaging and investigated SCN rhythmicity at a single cell resolution in free-behaving mice. We found that SCN neurons in vivo exhibited fast Ca2+ i spikes superimposed upon slow changes in baseline Ca2+ i levels. Both spikes and baseline followed a time-of-day modulation in many neurons, but independently from each other. Daily rhythms in basal Ca2+ i were highly coordinated, while spike activity from the same neurons peaked at multiple times of the light cycle, and unveiled clock-independent coactivity in neuron subsets. Hence, fast Ca2+ i spikes and slow changes in baseline Ca2+ i levels highlighted how multiple individual activity patterns could articulate within the temporal unity of the SCN cell network in vivo, and provided support for a multiplex neuronal code in the circadian pacemaker.

STING orchestrates the crosstalk between polyunsaturated fatty acid metabolism and inflammatory responses.

Concerted alteration of immune and metabolic homeostasis underlies several inflammation-related pathologies, ranging from metabolic syndrome to infectious diseases. Here, we explored the coordination of nucleic acid-dependent inflammatory responses and metabolic homeostasis. We reveal that the STING (stimulator of interferon genes) protein regulates metabolic homeostasis through inhibition of the fatty acid desaturase 2 (FADS2) rate-limiting enzyme in polyunsaturated fatty acid (PUFA) desaturation. STING ablation and agonist-mediated degradation increased FADS2-associated desaturase activity and led to accumulation of PUFA derivatives that drive thermogenesis. STING agonists directly activated FADS2-dependent desaturation, promoting metabolic alterations. PUFAs in turn inhibited STING, thereby regulating antiviral responses and contributing to resolving STING-associated inflammation. Thus, we have unveiled a negative regulatory feedback loop between STING and FADS2 that fine-tunes inflammatory responses. Our results highlight the role of metabolic alterations in human pathologies associated with aberrant STING activation and STING-targeting therapies.

Electroretinography of wild-type and Cry mutant mice reveals circadian tuning of photopic and mesopic retinal responses.

Attempts to understand circadian organization in the mammalian retina have concentrated increasingly on the mouse. However, rather little is known regarding circadian control of retinal light responses in this species. Here, the authors address this deficit using electroretinogram (ERG) recordings in C57BL/6 mice to evaluate rhythmicity in the wild-type retina and to identify the consequences of circadian clock loss in Cry1(- /-)Cry2(-/-) mice. They observe a circadian rhythm in the ERG waveform under light-adapted, cone-isolating conditions in wild-type mice, with b-wave speed and amplitude and the total power of oscillatory potentials all enhanced during the day. Wild types also exhibited a circadian dependence to ERG amplitude under dark-adapted conditions, but only when the flash stimulus was sufficiently bright to lie within the response range of cones. Cry1(-/ -)Cry2(-/-) mice lacked rhythmicity but retained superficially normal ERGs under all conditions suggesting that circadian clocks are dispensable for general retinal function. However, clock loss was associated with subtle abnormalities in retinal responses, with the amplitude of cone and mixed rod + cone ERGs constitutively enhanced. These data suggest that circadian clocks drive a fundamental fine-tuning of retinal pathways that is particularly apparent under conditions in which vision relies upon either cones alone or mixed rod + cone photoreception.

Cryptochrome-deficient mice lack circadian electrical activity in the suprachiasmatic nuclei.

The mammalian master clock driving circadian rhythmicity in physiology and behavior resides within the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. Circadian rhythms are generated by a set of clock genes via intertwined negative and positive autoregulatory transcription-translation feedback loops. The Cryptochrome 1 and 2 genes are indispensable for molecular core oscillator function, as evident from the arrhythmic wheel-running behavior and lack of rhythmic clock gene expression in mCry1/mCry2 double-mutant mice in constant darkness. In the present study, using real-time multiunit electrode activity recordings in hypothalamic slices, we show that SCN neurons from mCry-deficient mice kept in constant darkness lack circadian oscillations in firing patterns. This proves that cryptochromes, and thus an intact circadian clockwork, are prerequisites for circadian electrical activity in SCN neurons. Interestingly, when mCry-deficient mice were kept in normal light-dark conditions and SCN slices were prepared 2 hr after the beginning of the day, a single noncircadian peak in neuronal activity was detected. This light-induced rise in electrical activity of the SCN may explain why mCry-deficient mice lack the arrhythmic short bouts of wheel-running activity and instead show apparently normal behavior in normal day-night cycles.

Clock-dependent and system-driven oscillators interact in the suprachiasmatic nuclei to pace mammalian circadian rhythms.

Circadian clocks drive biological rhythms with a period of approximately 24 hours and keep in time with the outside world through daily resetting by environmental cues. While this external entrainment has been extensively investigated in the suprachiasmatic nuclei (SCN), the role of internal systemic rhythms, including daily fluctuations in core temperature or circulating hormones remains debated. Here, we show that lactating mice, which exhibit dampened systemic rhythms, possess normal molecular clockwork but impaired rhythms in both heat shock response gene expression and electrophysiological output in their SCN. This suggests that body rhythms regulate SCN activity downstream of the clock. Mathematical modeling predicts that systemic feedback upon the SCN functions as an internal oscillator that accounts for in vivo and ex vivo observations. Thus we are able to propose a new bottom-up hierarchical organization of circadian timekeeping in mammals, based on the interaction in the SCN between clock-dependent and system-driven oscillators.

Altered food-anticipatory activity rhythm in Cryptochrome-deficient mice.

In nocturnal rodents, restricted feeding to daytime (RF) causes feeding-associated diurnal locomotor activity that persists for the next 1-2 days when food is withheld. Along with this anticipatory behavior, the expression pattern of clock genes such as mPer1 and mPer2 changes from a nocturnal to diurnal pattern in the liver and cerebral cortex but not in the suprachiasmatic nucleus (SCN). Whether the molecular clockwork, in which mCry1 and mCry2 genes are essential components, is involved in food-anticipatory circadian rhythms is unknown. In this study, we investigated the impact of the absence of mCRY products upon the locomotion pattern induced by RF. RF caused an increase in daytime activity that lasted even for 2 days after food was withheld, in wild-type and mCry1-/-mCry2-/- mice. However, RF-induced activity was less stable and appeared more gradually in mutant mice. Similar results were obtained with mice housed under constant darkness or with SCN-lesioned wild-type and mutant mice. Our data reveal that mCry proteins are basically dispensable for food-entrainable oscillation. However, it is also important to note that mCry deficiency affects the stability and development of RF-induced anticipatory locomotor activity.

Assessment of lactotroph axis functionality in mice: longitudinal monitoring of PRL secretion by ultrasensitive-ELISA.

The pattern of prolactin (PRL) secretion depends on the physiological state. Due to insufficient detection sensitivity of existing assays, the precise description of these patterns in mice is lacking. We described an ultrasensitive ELISA assay that can detect mouse PRL in small fractions of whole blood, allowing longitudinal studies of PRL secretion profiles in freely moving mice. Over a 24-hour period, males displayed no oscillation in PRL levels, whereas virgin and lactating females showed large pulses. Peaks of PRL secretion reached 30-40 ng/mL in lactating female mice and rarely exceeded 10 ng/mL in virgin females. These pulses of PRL in lactating females were associated with suckling. The return of pups after an experimental 12-hour weaning induced a pulse of PRL release, reaching 100 ng/mL. This approach also enabled us to assess the inhibitory tone from hypothalamic dopamine neurons on PRL secretion. We used a dopamine D2 receptor antagonist to relieve pituitary lactotrophs from the tuberoinfundibular dopaminergic inhibitory tone and demonstrate a D2-induced PRL rise that can be used to evaluate both the secretory capacity of lactotrophs and the magnitude of the inhibitory tone on pituitary PRL release. We demonstrate that, although lactotroph function is altered to enhance chronic PRL output, their secretory response to acute stimulus is not modified during lactation and that chronic hyperprolactinemia is linked to a lower inhibitory tone. The combination of a sensitive PRL ELISA and administration of D2 receptor antagonist provide a unique opportunity to investigate the function and plasticity of the lactotroph axis in freely moving mice.

Electrical activity in endocrine pituitary cells in situ: a support for a multiple-function coding.

The anterior pituitary is an endocrine gland that controls basic body functions. Pituitary cell functioning depends on membrane excitability, which induces cytosolic calcium rises. Here, we reported the first identification of small-amplitude voltage fluctuations that controlled spike firing in endocrine cells recorded in situ. Three patterns of voltage fluctuations were distinguishable by their durations (1-100 s). These patterns could be ordered on top of each other, namely in response to secretagogues. Thus, pituitary endocrine cells express in situ a cell code in which small-amplitude voltage fluctuations lead to a multimodal arrangement of spike firing, which may finely tune calcium-dependent functions.

Light signalling in cryptochrome-deficient mice.

The mammalian master clock driving circadian rhythmicity in physiology, metabolism, and behaviour resides within the suprachiasmatic nuclei (SCN) of the anterior hypothalamus and is composed of intertwined negative and positive autoregulatory transcription-translation feedback loops. The Cryptochrome 1 and 2 gene products act in the negative feedback loop and are indispensable for molecular core oscillator function, as evident from the arrhythmic wheel running behaviour and absence of cyclic clock gene expression in mCry1/mCry2 double mutant mice in constant darkness. Recently, we have measured real-time multi-unit electrode activity recordings in hypothalamic slices from mCry-deficient mice kept in constant darkness and observed a complete lack of circadian oscillations in firing patterns. This proves that CRY proteins, and thus an intact circadian clock, are prerequisite for circadian rhythmicity in membrane excitability in SCN neurons. Strikingly, when mCry-deficient mice are housed in normal light-dark cycles, a single non-circadian peak in neuronal activity can be detected in SCN slices prepared two hours after the beginning of the day. This light-induced increase in electric activity of the SCN suggests that deletion of the mCry genes converts the core oscillator in an hour-glass-like timekeeper and may explain why in normal day-night cycles mCry-deficient mice show apparently normal behaviour.

Cognitive dysfunction, elevated anxiety, and reduced cocaine response in circadian clock-deficient cryptochrome knockout mice.

The circadian clock comprises a set of genes involved in cell-autonomous transcriptional feedback loops that orchestrate the expression of a range of downstream genes, driving circadian patterns of behavior. Cognitive dysfunction, mood disorders, anxiety disorders, and substance abuse disorders have been associated with disruptions in circadian rhythm and circadian clock genes, but the causal relationship of these associations is still poorly understood. In the present study, we investigate the effect of genetic disruption of the circadian clock, through deletion of both paralogs of the core gene cryptochrome (Cry1 and Cry2). Mice lacking Cry1 and Cry2 (Cry1(-/-)Cry2(-/-) ) displayed attenuated dark phase and novelty-induced locomotor activity. Moreover, they showed impaired recognition memory but intact fear memory. Depression-related behaviors in the forced swim test or sucrose preference tests were unaffected but Cry1(-/-)Cry2(-/-) mice displayed increased anxiety in the open field and elevated plus maze tests. Finally, hyperlocomotion and striatal phosphorylation of extracellular signal-regulated kinase (ERK) induced by a single cocaine administration are strongly reduced in Cry1(-/-)Cry2(-/-) mice. Interestingly, only some behavioral measures were affected in mice lacking either Cry1 or Cry2. Notably, recognition memory was impaired in both Cry1(-/-)Cry2(+/+) and Cry1(+/+)Cry2(-/-) mice. Moreover, we further observed elevated anxiety in Cry1(-/-)Cry2(+/+) and Cry1(+/+)Cry2(-/-) mice. Our data indicate that beyond their role in the control of circadian rhythm, cryptochrome genes have a direct influence in cognitive function, anxiety-related behaviors and sensitivity to psychostimulant drugs.

Modulation of the tyrosine kinase receptor Ret/glial cell-derived neurotrophic factor (GDNF) signaling: a new player in reproduction induced anterior pituitary plasticity?

During gestation, parturition, and lactation, the endocrine axis of the dam must continually adapt to ensure the continual and healthy development of offspring. The anterior pituitary gland, which serves as the endocrine interface between the brain and periphery, undergoes adaptations that contribute to regulation of the reproductive axis. Growth factors and their receptors are potential candidates for intrapituitary and paracrine factors to participate in the functional and anatomical plasticity of the gland. We examined the involvement of the growth factor glial cell-derived neurotrophic factor (GDNF) and its receptor tyrosine kinase rearranged during transfection (Ret) in the physiological functional and anatomical plasticity of the anterior pituitary gland. We found that variations in both expression and subcellular localization of Ret during gestation and lactation are temporally correlated with changes in pituitary gland function. We showed that Ret/GDNF signaling could endorse two different functional roles depending on the physiological status. At the end of lactation and after weaning, Ret was colocalized with markers of apoptosis. We found that Ret could therefore act as a physiological dependence receptor capable of inducing apoptosis in the absence of GDNF. In addition, we identified the follicullostellate cell as a probable source for intrapituitary GDNF and proposed GDNF as a potential physiological modulator of endocrine cell function. During all stages studied, we showed that acute application of GDNF to pituitary slices was able to modulate both positively and negatively intracellular calcium activity. Altogether our results implicate Ret/GDNF as a potent pleiotropic factor able to influence pituitary physiology during a period of high plasticity.

Circadian clock-coordinated hepatic lipid metabolism: only transcriptional regulation?

By regulating the metabolism of fatty acids, carbohydrates, and xenobiotic, the mammalian circadian clock plays a fundamental role on the liver physiology. At present, it is supposed that the circadian clock regulates metabolism mostly by regulating the expression of liver enzymes at the transcriptional level. However, recent evidences suggest that some signaling pathways synchronized by the circadian clock can also influence metabolism at a post-transcriptional level. In this context, we have recently shown that the circadian clock synchronizes the rhythmic activation of the IRE1alpha pathway in the endoplasmic reticulum. The absence of circadian clock perturbs this secondary clock, provokes deregulation of endoplasmic reticulum-localized enzymes, and leads to impaired lipid metabolism. We will describe here the additional pathways synchronized by the clock and discussed the influence of the circadian clock-controlled feeding rhythm on them.