GABA and Gi/o differentially control circadian rhythms and synchrony in clock neurons

Neurons in the mammalian suprachiasmatic nuclei (SCN) generate daily rhythms in physiology and behavior, but it is unclear how they maintain and synchronize these rhythms in vivo. We hypothesized that parallel signaling pathways in the SCN are required to synchronize rhythms in these neurons for coherent output. We recorded firing and clock-gene expression patterns while blocking candidate signaling pathways for at least 8 days. GABA(A) and GABA(B) antagonism increased circadian peak firing rates and rhythm precision of cultured SCN neurons, but G(i/o) did not impair synchrony or rhythmicity. In contrast, inhibiting G(i/o) with pertussis toxin abolished rhythms in most neurons and desynchronized the population, phenocopying the loss of vasoactive intestinal polypeptide (VIP). Daily VIP receptor agonist treatment restored synchrony and rhythmicity to VIP(-/-) SCN cultures during continuous GABA receptor antagonism but not during G(i/o) blockade. Pertussis toxin did not affect circadian cycling of the liver, suggesting that G(i/o) plays a specialized role in maintaining SCN rhythmicity. We conclude that endogenous GABA controls the amplitude of SCN neuronal rhythms by reducing daytime firing, whereas G(i/o) signaling suppresses nighttime firing, and it is necessary for synchrony among SCN neurons. We propose that G(i/o), not GABA activity, converges with VIP signaling to maintain and coordinate rhythms among SCN neurons.     

Lesions of suprachiasmatic nucleus efferents selectively affect rest-activity rhythm

The suprachiasmatic nucleus (SCN) of the hypothalamus controls circadian rhythms in behavioral, neuroendocrine and physiological functions. In this study, we test the hypothesis that caudal SCN efferents to the subparaventricular zone (SPVZ) control the rhythm in rest-activity (R-A) through projections on posterior hypothalamic area arousal systems (PHA). Small electrolytic lesions of the ventral SPVZ cause a selective loss of the circadian R-A rhythm, sparing the core body temperature rhythm. In contrast, large excitotoxic lesions of the posterior hypothalamic area (PHA) that effectively ablate populations of hypocretin and melanin concentrating hormone neurons projecting to cortex and subcortical arousal areas decrease R-A rhythm amplitude but do not disrupt circadian regulation. Since dorsomedial hypothalamic nucleus lesions have effects similar to PHA lesions (Chou, T.C., Scammell, T.E., Gooley, J.J., Gaus, S.E., Saper, C.B., Lu, J., 2003. Critical role of dorsomedial hypothalamic nucleus in a wide range of behavioral circadian rhythms. J. Neurosci. 23:10691-10702), these data support the view that the SPVZ is the principal relay nucleus for SCN signals to the multiple posterior hypothalamic arousal systems involved in generation of the R-A rhythm.     

Vasoactive intestinal polypeptide regulates dynamic changes in astrocyte morphometry: impact on gonadotropin-releasing hormone neurons

Recent studies suggest that astrocytes modulate the GnRH-induced LH surge. In particular, we have shown that the surface area of astrocytes that ensheath GnRH neurons exhibits diurnal rhythms. Vasoactive intestinal polypeptide (VIP) influences numerous aspects of astrocyte function in multiple brain regions and is a neurotransmitter in the suprachiasmatic nucleus (SCN) that affects GnRH neurons. The goals of this study were to: 1) assess whether astrocytes that surround GnRH neurons express VIP receptors, 2) determine the effects VIP suppression in the SCN on the morphometry of astrocytes surrounding GnRH neurons, and 3) assess whether this effect mimics aging-like changes in surface area of astrocytes. Young rats were ovariectomized (d 0), implanted with cannulae into the SCN (d 5), injected with VIP antisense (antioligo) or random sequence oligonucleotides, implanted with capsules containing 17beta-estradiol dissolved in oil (d 7), and perfused at 0300, 1400, and 1800 h (d 9). Brains were processed for immunocytochemistry. Our results demonstrate that astrocytes in close apposition to GnRH neurons express VIP receptors. Antioligo treatment blocked diurnal rhythms in surface area of astrocytes ensheathing GnRH neurons. The absence of diurnal rhythms resembles observations in middle-aged rats. Together these findings suggest that the ability of the VIP-containing neurons in the SCN to relay diurnal information to GnRH neurons may be by influencing dynamic changes in the morphometry of astrocytes that surround GnRH neurons. Furthermore, the absence of a VIP rhythm in aging animals may lead to altered GnRH activity via astrocyte-dependent mechanisms.     

Intrinsic,nondeterministic circadian rhythm generation in identified mammalian neurons

Circadian rhythms are modeled as reliable and self-sustained oscillations generated by single cells. The mammalian suprachiasmatic nucleus (SCN) keeps near 24-h time in vivo and in vitro, but the identity of the individual cellular pacemakers is unknown. We tested the hypothesis that circadian cycling is intrinsic to a unique class of SCN neurons by measuring firing rate or Period2 gene expression in single neurons. We found that fully isolated SCN neurons can sustain circadian cycling for at least 1 week. Plating SCN neurons at <100 cells/mm² eliminated synaptic inputs and revealed circadian neurons that contained arginine vasopressin (AVP) or vasoactive intestinal polypeptide (VIP) or neither. Surprisingly, arrhythmic neurons (nearly 80% of recorded neurons) also expressed these neuropeptides. Furthermore, neurons were observed to lose or gain circadian rhythmicity in these dispersed cell cultures, both spontaneously and in response to forskolin stimulation. In SCN explants treated with tetrodotoxin to block spike-dependent signaling, neurons gained or lost circadian cycling over many days. The rate of PERIOD2 protein accumulation on the previous cycle reliably predicted the spontaneous onset of arrhythmicity. We conclude that individual SCN neurons can generate circadian oscillations; however, there is no evidence for a specialized or anatomically localized class of cell-autonomous pacemakers. Instead, these results indicate that AVP, VIP, and other SCN neurons are intrinsic but unstable circadian oscillators that rely on network interactions to stabilize their otherwise noisy cycling.     

"Diabetes of the elderly" and type 2 diabetes in younger patients: possible role of the biological clock

The increased prevalence of type 2 diabetes in the aged has been recognized for a long time. Within the last decades, a growing number of younger subjects and even children are prone to develop type 2 diabetes. In both groups, aged and young, the biological clock, located in the suprachiasmatic nucleus of the hypothalamus (SCN) is malfunctioning as evidenced by disturbed sleep cycles and altered circadian rhythms. While elderly patients have an impaired function of the SCN due to the degeneration of neurons, we propose that in younger subjects the clock loses its "feeling" for internal and external rhythms caused by the modern lifestyle. Sleeping late and less coupled with constant metabolic excess alter both internal and external environmental stimuli to the brain. In response to these alterations, the rhythm of the biological clock is disrupted which may lead to the metabolic syndrome and type 2 diabetes.     

Development of circadian rhythmicity and light responsiveness in the rat suprachiasmatic nucleus: a study using the 2-deoxy[1-14C]glucose method.

The suprachiasmatic nucleus (SCN) of the hypothalamus is thought to play a critical role in circadian rhythm generation and entrainment to the light/dark cycle. In adult rats, the SCN shows a circadian rhythm in metabolic activity level as indicated by 2-deoxy[1-14C]glucose uptake. In the present study, the development of this rhythm was investigated. No diurnal difference in uptake was evident in fetal rats 1--2 days before birth. A significant diurnal difference in SCN 2-deoxyglucose uptake was preseno light at night increased the SCN metabolic levels. According to previous studies, on day 1 the SCN is poorly developed and contains few synapses. At this time the retinohypothalamic tract has not yet developed. We found progressive functional maturation of the SCN through day 21, when the rhythm and light responsiveness resembled those of adult rats.     

Sex hormone receptors are present in the human suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) is the clock of the brain that orchestrates circadian and circannual biological rhythms, such as the rhythms of hormones, body temperature, sleep and mood. These rhythms are frequently disturbed in menopause and even more so in dementia and can be restored in postmenopausal women by sex hormone replacement therapy (SHRT). Although it seems clear, both from clinical and experimental studies, that sex hormones influence circadian rhythms, it is not known whether this is by a direct or an indirect effect on the SCN. Therefore, using immunocytochemistry in the present study, we investigated whether the human SCN expresses sex hormone receptors in 5 premenopausal women and 5 young men. SCN neurons appeared to contain estrogen receptor-alpha (ERalpha), estrogen receptor-beta (ERbeta) and progesterone receptors. Median ratings of ER immunoreactivity per individual and per gender group revealed a statistically significantly stronger nuclear ERalpha expression pattern in female SCN neurons (p < 0.05). No significant sexual dimorphic tendency was observed for nuclear ERbeta (p > 0.1) and progesterone receptors (p > 0.7). These data seem to support previously reported functional and structural SCN differences in relation to sex and sexual orientation and indicate for the first time that estrogen and progesterone may act directly on neurons of the human biological clock. In addition, the present findings provide a potential neuroendocrine mechanism by which SHRT can act to improve or restore SCN-related rhythm disturbances, such as body temperature, sleep and mood.     

Melatonin rhythmicity: effect of age and Alzheimer's disease

The circadian rhythm of the pineal gland hormone, melatonin is generated within the hypothalamic suprachiasmatic nuclei (SCN), site of the circadian clock. The circadian clock and its output melatonin rhythm is synchronized to the 24h day by environmental light which is transmitted from the retina to the SCN primarily via the retinohypothalamic tract. Changes in both the amplitude and timing of the melatonin rhythm have been reported with aging in humans. Whether these age-related changes (reduced melatonin amplitude, earlier timing of melatonin rhythm) are a result of aging of the retina, the SCN clock, the pineal gland, their neural connections or a combination of some or all of these is not known. The fragmented sleep/wake patterns observed in the elderly and to a greater extent in patients with Alzheimer's disease have been shown to be partly related to an altered retina-SCN-pineal axis. Therapies designed to reinforce the circadian axis (for example, administration of melatonin or light) have been reported to alleviate the disturbed circadian rhythms and disrupted sleep. Future research needs to pinpoint the site(s) of age-related dysfunction so that therapies can be specifically tailored to correct the abnormality in addition to reinforcing any of the intact processes.     

Vasopressin messenger ribonucleic acid in supraoptic and suprachiasmatic nuclei: appearance and circadian regulation during development

The developmental appearance and regulation of hypothalamic vasopressin (prepropressophysin) mRNA was studied using quantitated in situ hybridization techniques. Vasopressin mRNA levels in the supraoptic nuclei were reliably detected on day 16 of gestation, while mRNA in the suprachiasmatic nuclei (SCN) was detectable on day 21. These developmental patterns correlate well with the immunohistochemical appearance of prepropressophysin translation products previously reported in these nuclei. A prominent day-night rhythm of vasopressin mRNA levels was evident in the SCN on day 21 of gestation; the rhythm was also present on postnatal days 2 and 11. No such rhythm was present in the supraoptic nuclei at any developmental stage examined. These results demonstrate regulated expression of the vasopressin gene during fetal life. Vasopressin mRNA levels in the SCN are already under specific circadian control when the nuclei are morphologically immature and lacking many of the connections found in adult animals.     

Expression of clock genes in the suprachiasmatic nucleus: Effect of environmental lighting conditions

The suprachiasmatic nucleus (SCN) is the anatomical substrate for the principal circadian clock coordinating daily rhythms in a vast array of behavioral and physiological responses. Individual SCN neurons are cellular oscillators and are organized into a multi-oscillator network following unique spatiotemporal patterns. The rhythms generated in the SCN are generally entrained to the environmental light dark cycle, which is the most salient cue influencing the network organization of the SCN. The neural network in the SCN is a heterogeneous structure, containing two major compartments identified by applying physiological and functional criteria, namely the retinorecipient core region and the highly rhythmic shell region. Changes in the environmental lighting condition are first detected and processed by the core region, and then conveyed to the rest of the SCN, leading to adaptive responses of the entire network. This review will focus on the studies that explore the responses of the SCN network by examining the expression of clock genes, under various lighting paradigms, such as acute light exposure, lighting schedules or exposure to different light durations. The results will be discussed under the framework of functionally distinct SCN sub regions and oscillator groups. The evidence presented here suggests that the environmental lighting conditions alter the spatiotemporal organization of the cellular oscillators within the SCN, which consequently affect the overt rhythms in behavior and physiology. Thus, information on how the SCN network elements respond to environmental cues is key to understanding the human health problems that stem from circadian rhythm disruption.     

The dorsomedial suprachiasmatic nucleus times circadian expression of Kiss1 and the luteinizing hormone surge

Ovulation in mammals is gated by a master circadian clock in the suprachiasmatic nucleus (SCN). GnRH neurons represent the converging pathway through which the brain triggers ovulation, but precisely how the SCN times GnRH neurons is unknown. We tested the hypothesis that neurons expressing kisspeptin, a neuropeptide coded by the Kiss1 gene and necessary for the activation of GnRH cells during ovulation, represent a relay station for circadian information that times ovulation. We first show that the circadian increase of Kiss1 expression, as well as the activation of GnRH cells, relies on intact ipsilateral neural input from the SCN. Second, by desynchronizing the dorsomedial (dm) and ventrolateral (vl) subregions of the SCN, we show that a clock residing in the dmSCN acts independently of the light-dark cycle, and the vlSCN, to time Kiss1 expression in the anteroventral periventricular nucleus of the hypothalamus and that this rhythm is always in phase with the LH surge. In addition, we show that although the timing of the LH surge is governed by the dmSCN, its amplitude likely depends on the phase coherence between the vlSCN and dmSCN. Our results suggest that whereas dmSCN neuronal oscillators are sufficient to time the LH surge through input to kisspeptin cells in the anteroventral periventricular nucleus of the hypothalamus, the phase coherence among dmSCN, vlSCN, and extra-SCN oscillators is critical for shaping it. They also suggest that female reproductive disorders associated with nocturnal shift work could emerge from the desynchronization between subregional oscillators within the master circadian clock.     

Prokineticin receptor 2 (Prokr2) is essential for the regulation of circadian behavior by the suprachiasmatic nuclei

The suprachiasmatic nucleus (SCN), the brain's principal circadian pacemaker, coordinates adaptive daily cycles of behavior and physiology, including the rhythm of sleep and wakefulness. The cellular mechanism sustaining SCN circadian timing is well characterized, but the neurochemical pathways by which SCN neurons coordinate circadian behaviors remain unknown. SCN transplant studies suggest a role for (unidentified) secreted factors, and one potential candidate is the SCN neuropeptide prokineticin 2 (Prok2). Prok2 and its cognate prokineticin receptor 2 (Prokr2/Gpcr73l1) are widely expressed in both the SCN and its neural targets, and Prok2 is light-regulated. Hence, they may contribute to cellular timing within the SCN, entrainment of the clock, and/or they may mediate circadian output. We show that a targeted null mutation of Prokr2 disrupts circadian coordination of the activity cycle and thermoregulation. Specifically, mice lacking Prokr2 lost precision in timing the onset of nocturnal locomotor activity; and under both a light/dark cycle and continuous darkness, there was a pronounced temporal redistribution of activity away from early to late circadian night. Moreover, the coherence of circadian behavior was significantly reduced, and nocturnal body temperature was depressed. Entrainment by light is not, however, dependent on Prokr2, and bioluminescence real-time imaging of organotypical SCN slices showed that the mutant SCN is fully competent as a circadian oscillator. We conclude that Prokr2 is not necessary for SCN cellular timekeeping or entrainment, but it is an essential link for coordination of circadian behavior and physiology by the SCN, especially in defining the onset and maintenance of circadian night.     

The central and basolateral nuclei of the amygdala exhibit opposite diurnal rhythms of expression of the clock protein Period2

There is considerable evidence that circadian rhythms in mammals can be modulated by emotional state, but how emotional state modulates specific circadian outputs is poorly understood. We analyzed the expression of the circadian clock protein Period2 (PER2) in three regions of the limbic forebrain known to play key roles in emotional regulation, the central nucleus of the amygdala (CEA), the basolateral amygdala (BLA), and the dentate gyrus (DG). We report here that cells in all three regions exhibit daily rhythms in expression of PER2 that are under the control of the master clock, the suprachiasmatic nucleus (SCN). The rhythm in the CEA and the rhythms in the BLA and DG are diametrically opposite in phase and are differentially affected by adrenalectomy. Adrenalectomy completely abolished the PER2 rhythm in the CEA but had no effect on the PER2 rhythms in the BLA and DG. We previously reported a rhythm in PER2 expression in the oval nucleus of the bed nucleus of the stria terminalis that is identical in phase and sensitivity to adrenalectomy to that found in the CEA. Together, these findings show that key structures of the limbic forebrain exhibit daily oscillations in clock gene expression that are controlled not only by input from the SCN but, importantly, by hormonal and neurochemical changes that normally accompany motivational and emotional states. Thus, cells within these areas are strategically positioned to integrate the inputs from the SCN and emotional states to modulate circadian rhythms downstream from the SCN clock.     

Vasoactive intestinal peptide and the mammalian circadian system

In mammals, the circadian oscillators that drive daily behavioral and endocrine rhythms are located in the hypothalamic suprachiasmatic nucleus (SCN). While the SCN is anatomically well-situated to receive and transmit temporal cues to the rest of the brain and periphery, there are many holes in our understanding of how this temporal regulation occurs. Unanswered questions include how cell autonomous circadian oscillations within the SCN remain synchronized to each other as well as communicate temporal information to downstream targets. In recent years, it has become clear that neuropeptides are critically involved in circadian timekeeping. One such neuropeptide, vasoactive intestinal peptide (VIP), defines a cell population within the SCN and is likely used as a signaling molecule by these neurons. Converging lines of evidence suggest that the loss of VIP or its receptor has a major influence on the ability of the SCN neurons to generate circadian oscillations as well as synchronize these cellular oscillations. VIP, acting through the VPAC(2) receptor, exerts these effects in the SCN by activating intracellular signaling pathways and, consequently, modulating synaptic transmission and intrinsic membrane currents. Anatomical evidence suggests that these VIP expressing neurons connect both directly and indirectly to endocrine and other output targets. Striking similarities exist between the role of VIP in mammals and the role of Pigment Dispersing Factor (PDF), a functionally related neuropeptide, in the Drosophila circadian system. Work in both mammals and insects suggests that further research into neuropeptide function is necessary to understand how circadian oscillators work as a coordinated system to impose a temporal structure on physiological processes within the organism.     

Diurnal variation in rat liver thyroid hormone receptor (TR)-alpha messenger ribonucleic acid (mRNA) is dependent on the biological clock in the suprachiasmatic nucleus, whereas diurnal variation of TR beta 1 mRNA is modified by food intake

Previous studies have shown a diurnal variation of certain isoforms of thyroid hormone receptors (TR) in rat liver. The genesis of these diurnal changes is still unknown. To clarify whether the biological clock, located in the hypothalamic suprachiasmatic nucleus (SCN), is involved, we made selective SCN lesions. Rats with an SCN lesion lost their circadian rhythm of plasma corticosterone and TSH when compared with intact animals. TR alpha 1 and TR alpha 2 mRNA expression of control rats was higher in the light period than in the dark period; changes that were abolished in the rats with SCN lesions. In contrast, liver TR beta 1 mRNA of intact rats showed a diurnal variation that failed to reach statistical significance. To evaluate whether these effects could be explained indirectly by the disappearance of rhythmic feeding behavior in rats with SCN lesions, we performed a second experiment in which otherwise intact animals were subjected to a regular feeding (RF) schedule, with one meal every 4 h. When compared with rats with free access to food, RF only affected TR beta 1 mRNA expression and had no effect on the diurnal changes in TR alpha 1 and TR alpha 2. We conclude that liver TR beta 1 expression is most clearly affected by food intake. Diurnal changes in liver TR alpha 1 and TR alpha 2 are controlled by the biological clock in the SCN but not via changes in the daily rhythm of food intake. The findings may have physiological relevance for diurnal variation of T(3)-dependent gene expression, which is supported by a diurnal variation in the expression of the 5'-deiodinase gene.     

Heterogeneity of rhythmic suprachiasmatic nucleus neurons: Implications for circadian waveform and photoperiodic encoding

Circadian rhythms in neuronal ensemble, subpopulations, and single unit activity were recorded in the suprachiasmatic nuclei (SCN) of rat hypothalamic slices. Decomposition of the ensemble pattern revealed that neuronal subpopulations and single units within the SCN show surprisingly short periods of enhanced electrical activity of approximately 5 h and show maximal activity at different phases of the circadian cycle. The summed activity accounts for the neuronal ensemble pattern of the SCN, indicating that circadian waveform of electrical activity is a composed tissue property. The recorded single unit activity pattern was used to simulate the responsiveness of SCN neurons to different photoperiods. We inferred predictions on changes in peak width, amplitude, and peak time in the multiunit activity pattern and confirmed these predictions with hypothalamic slices from animals that had been kept in a short or long photoperiod. We propose that the animals' ability to code for day length derives from plasticity in the neuronal network of oscillating SCN neurons.