Andries Kalsbeek

Pineal clock gene oscillation is disturbed in Alzheimer’s disease, due to functional disconnection from the “master clock”

The suprachiasmatic nucleus (SCN) is the “master clock” of the mammalian brain. It coordinates the peripheral clocks in the body, including the pineal clock that receives SCN input via a multisynaptic noradrenergic pathway. Rhythmic pineal melatonin production is disrupted in Alzheimers disease (AD). Here we show that the clock genes hBmal1, hCry1, and hPer1 were rhythmically expressed in the pineal of controls (Braak 0). Moreover, hPer1 and hβ1‐adrenergic receptor (hβ1‐ADR) mRNA were positively correlated and showed a similar daily pattern. In contrast, in both preclinical (Braak I‐II) and clinical AD patients (Braak V‐VI), the rhythmic expression of clock genes was lost as well as the correlation between hPer1 and hβ1‐ADR mRNA. Intriguingly, hCry1 mRNA was increased in clinical AD. These changes are probably due to a disruption of the SCN control, as they were mirrored in the rat pineal deprived of SCN control. Indeed, a functional disruption of the SCN was observed from the earliest AD stages onward, as shown by decreased vasopressin mRNA, a clock‐controlled major output of the SCN. Thus, a functional disconnection between the SCN and the pineal from the earliest AD stage onward could account for the pineal clock gene changes and underlie the circadian rhythm disturbances in AD.—Wu, Y‐H., Fischer, D. F., Kalsbeek, A., Garidou‐Boof, M‐L., van der Vliet, J., van Heijningen, C., Liu, R‐Y., Zhou, J‐N., Swaab, D. F. Pineal clock gene oscillation is disturbed in Alzheimers disease, due to functional disconnection from the “master clock.” FASEB J. 20, E1171–E1180 (2006)

The Suprachiasmatic Nucleus Controls Circadian Energy Metabolism and Hepatic Insulin Sensitivity

Disturbances in the circadian system are associated with the development of type 2 diabetes mellitus. Here, we studied the direct contribution of the suprachiasmatic nucleus (SCN), the central pacemaker in the circadian system, in the development of insulin resistance. Exclusive bilateral SCN lesions in male C57Bl/6J mice, as verified by immunochemistry, showed a small but significant increase in body weight (+17%), which was accounted for by an increase in fat mass. In contrast, mice with collateral damage to the ventromedial hypothalamus and paraventricular nucleus showed severe obesity and insulin resistance. Mice with exclusive SCN ablation revealed a loss of circadian rhythm in activity, oxygen consumption, and food intake. Hyperinsulinemic–euglycemic clamp analysis 8 weeks after lesioning showed that the glucose infusion rate was significantly lower in SCN lesioned mice compared with sham-operated mice (−63%). Although insulin potently inhibited endogenous glucose production (−84%), this was greatly reduced in SCN lesioned mice (−7%), indicating severe hepatic insulin resistance. Our data show that SCN malfunctioning plays an important role in the disturbance of energy balance and suggest that an absence of central clock activity, in a genetically intact animal, may lead to the development of insulin resistance.

GABA release from suprachiasmatic nucleus terminals is necessary for the light-induced inhibition of nocturnal melatonin release in the rat

The daily rhythm of melatonin production in the mammalian pineal is driven by the endogenous circadian pacemaker in the suprachiasmatic nuclei. The major release period of melatonin is closely linked to the dark phase of the 24-h day/night cycle. Environmental light will affect melatonin release in two ways: (i) it entrains the rhythm of the circadian oscillator; and (ii) it causes an acute suppression of nocturnal melatonin release. These two effects of light are both mediated by the suprachiasmatic nucleus and enable the pineal gland to convey information about day length to the reproductive system through changes in melatonin levels. Glutamate is currently believed to be the major transmitter in the retinal ganglion cell fibers reaching the suprachiasmatic nucleus. At present no information is available, however, about the transmitter(s) implicated in the further propagation, i.e. from the suprachiasmatic nucleus onwards, of the light information. In the present study we provide evidence that the endogenous release of GABA from suprachiasmatic nucleus terminals is implicated in the further transmission of light information to the pineal gland. Bilateral administration of the GABA-antagonist bicuculline to hypothalamic target areas of the suprachiasmatic nucleus completely prevents the inhibitory effect of nocturnal light on melatonin secretion and the present study thus documents that retina-mediated photic activation of suprachiasmatic nucleus neurons induces the release of GABA from efferent suprachiasmatic nucleus nerve terminals, resulting in an inhibition of melatonin release by the pineal gland. Together with our previous (electro)physiological data these results identify GABA as an important mediator of rapid synaptic transmission of suprachiasmatic nucleus output to its target areas.

Effects of Nocturnal Light on (Clock) Gene Expression in Peripheral Organs: A Role for the Autonomic Innervation of the Liver

Background The biological clock, located in the hypothalamic suprachiasmatic nucleus (SCN), controls the daily rhythms in physiology and behavior. Early studies demonstrated that light exposure not only affects the phase of the SCN but also the functional activity of peripheral organs. More recently it was shown that the same light stimulus induces immediate changes in clock gene expression in the pineal and adrenal, suggesting a role of peripheral clocks in the organ-specific output. In the present study, we further investigated the immediate effect of nocturnal light exposure on clock genes and metabolism-related genes in different organs of the rat. In addition, we investigated the role of the autonomic nervous system as a possible output pathway of the SCN to modify the activity of the liver after light exposure. Methodology and Principal Findings First, we demonstrated that light, applied at different circadian times, affects clock gene expression in a different manner, depending on the time of day and the organ. However, the changes in clock gene expression did not correlate in a consistent manner with those of the output genes (i.e., genes involved in the functional output of an organ). Then, by selectively removing the autonomic innervation to the liver, we demonstrated that light affects liver gene expression not only via the hormonal pathway but also via the autonomic input. Conclusion Nocturnal light immediately affects peripheral clock gene expression but without a clear correlation with organ-specific output genes, raising the question whether the peripheral clock plays a “decisive” role in the immediate (functional) response of an organ to nocturnal light exposure. Interestingly, the autonomic innervation of the liver is essential to transmit the light information from the SCN, indicating that the autonomic nervous system is an important gateway for the SCN to cause an immediate resetting of peripheral physiology after phase-shift inducing light exposures.

GABA Receptors in the Region of the Dorsomedial Hypothalamus of Rats Are Implicated in the Control of Melatonin and Corticosterone Release

Recently, anatomical evidence was presented that the mammalian circadian clock located in the suprachiasmatic nuclei (SCN) may utilize GABA to transmit diurnal information to the dorsomedial hypothalamus (DMH). The present study provides further physiological evidence for the involvement of this GABAergic projection in the regulation of diurnal rhythms. Infusion of the GABA agonist muscimol in the region of the DMH completely blocked the daily increase of plasma melatonin during darkness and reduced sympathetic output in the pineal gland resulting in lower pineal melatonin production, as measured with transpineal microdialysis. Further experiments in SCN-lesioned animals indicated that the origin of this inhibitory input to the DMH is indeed the SCN. The results of this study imply that the SCN can influence the sympathetic outflow of the hypothalamus through its GABA-containing projection. Furthermore, the present results probably explain the previously reported strong inhibitory effect of benzodiazepines on plasma melatonin in both animals and humans.

Central effects of thyronamines on glucose metabolism in rats

Thyronamines are naturally occurring, chemical relatives of thyroid hormone. Systemic administration of synthetic 3-iodothyronamine (T(1)AM) and - to a lesser extent - thyronamine (T(0)AM), leads to acute bradycardia, hypothermia, decreased metabolic rate, and hyperglycemia. This profile led us to hypothesize that the central nervous system is among the principal targets of thyronamines. We investigated whether a low dose i.c.v. infusion of synthetic thyronamines recapitulates the changes in glucose metabolism that occur following i.p. thyronamine administration. Plasma glucose, glucoregulatory hormones, and endogenous glucose production (EGP) using stable isotope dilution were monitored in rats before and 120 min after an i.p. (50 mg/kg) or i.c.v. (0.5 mg/kg) bolus infusion of T(1)AM, T(0)AM, or vehicle. To identify the peripheral effects of centrally administered thyronamines, drug-naive rats were also infused intravenously with low dose (0.5 mg/kg) thyronamines. Systemic T(1)AM rapidly increased EGP and plasma glucose, increased plasma glucagon, and corticosterone, but failed to change plasma insulin. Compared with i.p.-administered T(1)AM, a 100-fold lower dose administered centrally induced a more pronounced acute EGP increase and hyperglucagonemia while plasma insulin tended to decrease. Both systemic and central infusions of T(0)AM caused smaller increases in EGP, plasma glucose, and glucagon compared with T(1)AM. Neither T(1)AM nor T(0)AM influenced any of these parameters upon low dose i.v. administration. We conclude that central administration of low-dose thyronamines suffices to induce the acute alterations in glucoregulatory hormones and glucose metabolism following systemic thyronamine infusion. Our data indicate that thyronamines can act centrally to modulate glucose metabolism.

Opposite actions of hypothalamic vasopressin on circadian corticosterone rhythm in nocturnal versus diurnal species

Relatively little is known about the function of the biological clock and its efferent pathways in diurnal species, despite the fact that its major transmitters and neuronal connections are also conserved in humans. The mammalian biological clock is located in the hypothalamic suprachiasmatic nuclei (SCN). Several lines of evidence suggest that the activity cycle of the SCN itself is similar in nocturnal and diurnal mammals. Previously, we showed that, in the rat, vasopressin (VP) derived from the SCN has a strong inhibitory effect on the release of adrenal corticosterone and is an important component in the generation of a daily rhythm in plasma corticosterone concentrations. In the present study we investigated the role of VP in the control of the daily corticosterone rhythm in a diurnal rodent, i.e. Arvicanthis ansorgei. Contrary to our previous (rat) results, VP administered to the hypothalamic paraventricular nucleus in A. ansorgei had a stimulatory effect on the release of corticosterone. Moreover, both the morning and evening rise in corticosterone were blocked by the administration of a VP receptor antagonist. These results show that with regard to the circadian control of the corticosterone rhythm in diurnal and nocturnal rodents, temporal information is carried along the same pathway from the SCN to its target areas, but the response of the target area may be quite different. We propose that the reversed response to VP is due to a change in the phenotype of the target neurons that are contacted by the SCN efferents, i.e. glutamatergic instead of γ‐aminobutyric acid (GABA)ergic.

A circulating ghrelin mimetic attenuates light-induced phase delay of mice and light-induced Fos expression in the suprachiasmatic nucleus of rats

Anatomical evidence suggests that the ventromedial arcuate nucleus (vmARC) is a route for circulating hormonal communications to the suprachiasmatic nucleus (SCN). Whether this vmARC–SCN connection is involved in the modulation of circadian activity of the SCN is not yet known. We recently demonstrated, in rats, that intravenous (i.v.) injection of a ghrelin mimetic, GHRP‐6, during the daytime activated neurons in the vmARC and reduced the normal endogenous daytime Fos expression in the SCN. In the present study we show that i.v. administration of GHRP‐6 decreases light‐induced Fos expression at ZT13 in the rat SCN by 50%, indicating that light‐induced changes in the SCN Fos expression can also be reduced by GHRP‐6. Because it is difficult to study light‐induced phase changes in rats, we examined the functional effects of GHRP‐6 on light‐induced phase shifts in mice and demonstrated that peripherally injected GHRP‐6 attenuates light‐induced phase delays at ZT13 by 45%. However, light‐induced Fos expression in the mice SCN was not blocked by GHRP‐6. These results illustrate that acute stimulation of the ghrelinergic system may modulate SCN activity, but that its effect on light‐induced phase shifts and Fos expression in the SCN might be species related.

Standards of evidence in chronobiology: critical review of a report that restoration of Bmal1 expression in the dorsomedial hypothalamus is sufficient to restore circadian food anticipatory rhythms in Bmal1-/- mice

Daily feeding schedules generate food anticipatory rhythms of behavior and physiology that exhibit canonical properties of circadian clock control. The molecular mechanisms and location of food-entrainable circadian oscillators hypothesized to control food anticipatory rhythms are unknown. In 2008, Fuller et al reported that food-entrainable circadian rhythms are absent in mice bearing a null mutation of the circadian clock gene Bmal1 and that these rhythms can be rescued by virally-mediated restoration of Bmal1 expression in the dorsomedial nucleus of the hypothalamus (DMH) but not in the suprachiasmatic nucleus (site of the master light-entrainable circadian pacemaker). These results, taken together with controversial DMH lesion results published by the same laboratory, appear to establish the DMH as the site of a Bmal1-dependent circadian mechanism necessary and sufficient for food anticipatory rhythms. However, careful examination of the manuscript reveals numerous weaknesses in the evidence as presented. These problems are grouped as follows and elaborated in detail: 1. data management issues (apparent misalignments of plotted data), 2. failure of evidence to support the major conclusions, and 3. missing data and methodological details. The Fuller et al results are therefore considered inconclusive, and fail to clarify the role of either the DMH or Bmal1 in the expression of food-entrainable circadian rhythms in rodents.

In vivo evidence for a controlled offset of melatonin synthesis at dawn by the suprachiasmatic nucleus in the rat.

The daily rhythm of melatonin synthesis in the rat pineal gland is controlled by the central biological clock, located in the suprachiasmatic nucleus (SCN), via a multi-synaptic pathway involving, successively, neurones of the paraventricular nucleus of the hypothalamus (PVN), sympathetic preganglionic neurones of the intermediolateral cell column of the spinal cord, and norepinephrine containing sympathetic neurones of the superior cervical ganglion. Recently, we showed that, in the rat, the SCN uses a combination of daytime inhibitory and nighttime stimulatory signals toward the PVN-pineal pathway in order to control the daily rhythm of melatonin synthesis, GABA being responsible for the daytime inhibitory message and glutamate for the nighttime stimulation. The present study was initiated to further check this concept, and to investigate the involvement of the inhibitory SCN output in the early morning circadian decline of melatonin release, with the hypothesis that, at dawn, the increased release of GABA onto pre-autonomic PVN neurones results in a diminished norepinephrine stimulation of the pineal, and ultimately an arrest of melatonin release. First, we established that prolonged norepinephrine stimulation of the pineal gland was indeed sufficient to prevent the early morning decline of melatonin release. Blockade of GABA-ergic signaling in the PVN at dawn could not prevent the early morning decline of melatonin completely. Therefore, these results show that an increased GABAergic inhibition of the PVN neurones that control the sympathetic innervation of the pineal gland, at dawn, is not sufficient to explain the early morning decline of melatonin release.