A Network of (Autonomic) Clock Outputs

The circadian clock in the suprachiasmatic nuclei (SCN) is composed of thousands of oscillator neurons, each of which is dependent on the cell‐autonomous action of a defined set of circadian clock genes. A major question is still how these individual oscillators are organized into a biological clock producing a coherent output that is able to time all the different daily changes in behavior and physiology. We investigated which anatomical connections and neurotransmitters are used by the biological clock to control the daily release pattern of a number of hormones. The picture that emerged shows projections contacting target neurons in the medial hypothalamus surrounding the SCN. The activity of these pre‐autonomic and neuro‐endocrine target neurons is controlled by differentially timed waves of, among others, vasopressin, GABA, and glutamate release from SCN terminals. Together our data indicate that, with regard to the timing of their main release period within the light‐dark (LD) cycle, at least 4 subpopulations of SCN neurons should be discerned. The different subgroups do not necessarily follow the phenotypic differences among SCN neurons. Thus, different subgroups can be found within neuron populations containing the same neurotransmitter. Remarkably, a similar distinction of 4 differentially timed subpopulations of SCN neurons was recently also discovered in experiments determining the temporal patterns of rhythmicity in individual SCN neurons by way of the electrophysiology or clock gene expression. Moreover, the specialization of the SCN may go as far as a single body structure; i.e., the SCN seems to contain neurons that specifically target the liver, pineal, and adrenal.     

Output pathways of the mammalian suprachiasmatic nucleus: coding circadian time by transmitter selection and specific targeting

Every day, we experience profound changes in our mental and physical condition as body and brain alternate between states of high activity during the waking day and rest during night-time sleep. The fundamental evolutionary adaptation to these profound daily changes in our physiological state is an endogenous 24-h clock. This biological clock enables us to prepare ourselves to these daily changes, instead of only being able to show a passive and delayed response. During the past decade, enormous progress has been made in determining possible molecular components of the biological clock. An important question remains, however, regarding how the rhythmic signal from the biological clock is spread throughout the body to control its physiology and behavior. Indeed, ultimately, the only raison d'etre for the biological clock is its output (Green 1998). In the present review, we propose that the main mechanism for the spreading time-of-day information throughout the body consists of different circadian waves of suprachiasmatic nucleus (SCN) transmitter release, directed to a restricted number of specific SCN target areas, and affecting both neuroendocrine mechanisms and the peripheral autonomic nervous system.     

Melatonin as a time-meaningful signal in circadian organization of immune response

Melatonin is synthesized and secreted during the dark period of the light/dark cycle. The rhythmic nocturnal melatonin secretion is directly generated by the circadian clock, located within the suprachiasmatic nuclei in mammals and is entrained to a 24-hour period by the light-dark cycle. The periodic secretion of melatonin may be used as a circadian mediator to any system that can 'read' the message. Melatonin seems to act as an arm of the circadian clock, giving a time-related signal to a number of body functions; one of these, the circadian organization of the defense of the organism, is discussed in some detail as an example.     

Signals from the Brainstem Sleep/Wake Centers Regulate Behavioral Timing via the Circadian Clock

Sleep-wake cycling is controlled by the complex interplay between two brain systems, one which controls vigilance state, regulating the transition between sleep and wake, and the other circadian, which communicates time-of-day. Together, they align sleep appropriately with energetic need and the day-night cycle. Neural circuits connect brain stem sites that regulate vigilance state with the suprachiasmatic nucleus (SCN), the master circadian clock, but the function of these connections has been unknown. Coupling discrete stimulation of pontine nuclei controlling vigilance state with analytical chemical measurements of intra-SCN microdialysates in mouse, we found significant neurotransmitter release at the SCN and, concomitantly, resetting of behavioral circadian rhythms. Depending upon stimulus conditions and time-of-day, SCN acetylcholine and/or glutamate levels were augmented and generated shifts of behavioral rhythms. These results establish modes of neurochemical communication from brain regions controlling vigilance state to the central circadian clock, with behavioral consequences. They suggest a basis for dynamic integration across brain systems that regulate vigilance states, and a potential vulnerability to altered communication in sleep disorders.     

A Network of (Autonomic) Clock Outputs

The circadian clock in the suprachiasmatic nuclei (SCN) is composed of thousands of oscillator neurons, each dependent on the cell‐autonomous action of a defined set of circadian clock genes. A major question is still how these individual oscillators are organized into a biological clock that produces a coherent output capable of timing all the different daily changes in behavior and physiology. We investigated which anatomical connections and neurotransmitters are used by the biological clock to control the daily release pattern of a number of hormones. The picture that emerged shows projections contacting target neurons in the medial hypothalamus surrounding the SCN. The activity of these pre‐autonomic and neuro‐endocrine target neurons is controlled by differentially timed waves of vasopressin, GABA, and glutamate release from SCN terminals, among other factors. Together our data indicate that, with regard to the timing of their main release period within the LD cycle, at least four subpopulations of SCN neurons should be discernible. The different subgroups do not necessarily follow the phenotypic differences among SCN neurons. Thus, different subgroups can be found within neuron populations containing the same neurotransmitter. Remarkably, a similar distinction of four differentially timed subpopulations of SCN neurons was recently also discovered in experiments determining the temporal patterns of rhythmicity in individual SCN neurons by way of the electrophysiology or clock gene expression. Moreover, the specialization of the SCN may go as far as a single body structure, i.e., the SCN seems to contain neurons that specifically target the liver, pineal gland, and adrenal gland.     

Melatonin and biological rhythms

Circadian and seasonal rhythms are a fundamental feature of all living organisms. The functional mechanism involved is built around internal biological clock(s) and the hormone melatonin (Mel) is one of its critical components. Although numerous other sources have been identified (retina, harderian gland, gut), in vertebrates Mel is primarily produced by the pineal gland during the dark period of the light-dark cycle. This rhythmic Mel is generated directly by circadian clock(s). The Mel rhythm is thus an important efferent hormonal signal from the clock. The periodic secretion of Mel might thus be used as a circadian mediator of a system that can 'read' the message.The duration of the nocturnal Mel production is directly proportional to the length of the dark period. It is through these changes in duration that the brain integrates the photoperiodic information. In essence, the Mel rhythm appears to be an endocrine code of the environmental light-dark cycle conveying photic information that is used by organisms for both circadian and seasonal temporal organization. The major question arising from this effect of Mel concerns it precise mechanism of action. From the data reported in the present minireview, it appears that the photoneuroendocrine mechanism is not fundamentally different in vertebrates at least as far as the role of Mel is concerned.     

Amplitude of the SCN clock enhanced by the behavioral activity rhythm

Circadian rhythms are regulated by the suprachiasmatic nucleus (SCN), a small structure at the base of the hypothalamus. While light effects on the SCN are well established, little is known of behavioral effects. This study elucidates direct modulating action of behavioral activity on the SCN by use of in vivo electrophysiology recordings, assessments of general locomotor behavior, and video-tracking of mice. The results show suppression of SCN neuronal activity by spontaneous behavior, the magnitude being dependent on the intensity, duration and type of behavioral activity. The suppression was moderate (32% of circadian amplitude) for low-intensity behavior and considerable (59%) for locomotor activity. Mild manipulation of the animals had reversed effects on the SCN indicating that different mechanisms are involved in the regulatory effect of spontaneous versus induced activity. The results indicate that exercise at the proper time of the cycle can boost the amplitude of the rhythm of the SCN clock itself. This has potentially beneficial effects for other rhythmic functions that are under the control of the SCN.     

A network of (autonomic) clock outputs

The circadian clock in the suprachiasmatic nuclei (SCN) is composed of thousands of oscillator neurons, each of which is dependent on the cell-autonomous action of a defined set of circadian clock genes. A major question is still how these individual oscillators are organized into a biological clock producing a coherent output that is able to time all the different daily changes in behavior and physiology. We investigated which anatomical connections and neurotransmitters are used by the biological clock to control the daily release pattern of a number of hormones. The picture that emerged shows projections contacting target neurons in the medial hypothalamus surrounding the SCN. The activity of these pre-autonomic and neuro-endocrine target neurons is controlled by differentially timed waves of, among others, vasopressin, GABA, and glutamate release from SCN terminals. Together our data indicate that, with regard to the timing of their main release period within the light-dark (LD) cycle, at least 4 subpopulations of SCN neurons should be discerned. The different subgroups do not necessarily follow the phenotypic differences among SCN neurons. Thus, different subgroups can be found within neuron populations containing the same neurotransmitter. Remarkably, a similar distinction of 4 differentially timed subpopulations of SCN neurons was recently also discovered in experiments determining the temporal patterns of rhythmicity in individual SCN neurons by way of the electrophysiology or clock gene expression. Moreover, the specialization of the SCN may go as far as a single body structure; i.e., the SCN seems to contain neurons that specifically target the liver, pineal, and adrenal.     

Food ingestion and circadian rhythmicity

Feeding is organised within the 24-h of the light - dark (LD) cycle. Food is ingested in a circadian manner in nature and in laboratory animals kept under constant conditions. The circadian rhythmicity in food ingestion is driven by a biological clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus. The circadian organisation of food ingestion not only allows animals to live in harmony with their environment but food intake could also act as a zeitgeber for other rhythmic functions. Lesions in the area of the SCN result in the loss of most rhythmic functions as well as to a disrupted circadian rhythmicity of food and water intake. These findings, together with observations from daytime feeding experiments conducted in nocturnal animals, suggest that food intake may serve as a temporal signal for some peripheral organs to oscillate in phase with the SCN. This paper overviews and discusses how food intake interacts with the circadian system.     

A network of (autonomic) clock outputs

The circadian clock in the suprachiasmatic nuclei (SCN) is composed of thousands of oscillator neurons, each dependent on the cell-autonomous action of a defined set of circadian clock genes. A major question is still how these individual oscillators are organized into a biological clock that produces a coherent output capable of timing all the different daily changes in behavior and physiology. We investigated which anatomical connections and neurotransmitters are used by the biological clock to control the daily release pattern of a number of hormones. The picture that emerged shows projections contacting target neurons in the medial hypothalamus surrounding the SCN. The activity of these pre-autonomic and neuro-endocrine target neurons is controlled by differentially timed waves of vasopressin, GABA, and glutamate release from SCN terminals, among other factors. Together our data indicate that, with regard to the timing of their main release period within the LD cycle, at least four subpopulations of SCN neurons should be discernible. The different subgroups do not necessarily follow the phenotypic differences among SCN neurons. Thus, different subgroups can be found within neuron populations containing the same neurotransmitter. Remarkably, a similar distinction of four differentially timed subpopulations of SCN neurons was recently also discovered in experiments determining the temporal patterns of rhythmicity in individual SCN neurons by way of the electrophysiology or clock gene expression. Moreover, the specialization of the SCN may go as far as a single body structure, i.e., the SCN seems to contain neurons that specifically target the liver, pineal gland, and adrenal gland.     

Emerging links between the biological clock and the DNA damage response

For life forms to survive, they must adapt to their environmental conditions. One such factor that impacts on both prokaryotic and eukaryotic organisms is the light–dark cycle, a consequence of planetary rotation in relation to our sun. In mammals, the daily light cycle has affected the regulation of many cellular processes such as sleep–wake and calorific intake activities, hormone secretion, blood pressure and immune system responses. Such rhythmic behaviour is the consequence of circadian rhythm/biological clock (BC) systems which are controlled in a light stimulus-dependent manner by a master clock called the suprachiasmatic nucleus (SCN) situated within the anterior hypothalamus. Peripheral clocks located in other organs such as the liver and kidneys relay signals from the SCN, which ultimately leads to tightly controlled expression of several protein families that in turn act on a broad range of cellular functions. Work in lower organisms has demonstrated a link between aging processes and BC factors, and studies in both animal models and clinical trials have postulated a role for certain BC-associated proteins in tumourigenesis and cancer progression. Recent exciting data reported within the last year or so have now established a molecular link between specific BC proteins and factors that control the mammalian cell cycle and DNA damage checkpoints. This mini review will focus on these discoveries and emphasise how such BC proteins may be involved, through their interplay with cell cycle/DNA damage response pathways, in the development of human disease such as cancer.     

Clock Genes and Behavioral Responses to Light Are Altered in a Mouse Model of Diabetic Retinopathy

There is increasing evidence that melanopsin-expressing ganglion cells (ipRGCs) are altered in retinal pathologies. Using a streptozotocin-induced (STZ) model of diabetes, we investigated the impact of diabetic retinopathy on non-visual functions by analyzing ipRGCs morphology and light-induced c-Fos and Period 1–2 clock genes in the central clock (SCN). The ability of STZ-diabetic mice to entrain to light was challenged by exposure animals to 1) successive light/dark (LD) cycle of decreasing or increasing light intensities during the light phase and 2) 6-h advance of the LD cycle. Our results show that diabetes induces morphological changes of ipRGCs, including soma swelling and dendritic varicosities, with no reduction in their total number, associated with decreased c-Fos and clock genes induction by light in the SCN at 12 weeks post-onset of diabetes. In addition, STZ-diabetic mice exhibited a reduction of overall locomotor activity, a decrease of circadian sensitivity to light at low intensities, and a delay in the time to re-entrain after a phase advance of the LD cycle. These novel findings demonstrate that diabetes alters clock genes and behavioral responses of the circadian timing system to light and suggest that diabetic patients may show an increased propensity for circadian disturbances, in particular when they are exposed to chronobiological challenges.     

Temporal phasing of locomotor activity, heart rate rhythmicity, and core body temperature is disrupted in VIP receptor 2-deficient mice

Neurons of the brain's biological clock located in the hypothalamic suprachiasmatic nucleus (SCN) generate circadian rhythms of physiology (core body temperature, hormone secretion, locomotor activity, sleep/wake, and heart rate) with distinct temporal phasing when entrained by the light/dark (LD) cycle. The neuropeptide vasoactive intestinal polypetide (VIP) and its receptor (VPAC2) are highly expressed in the SCN. Recent studies indicate that VIPergic signaling plays an essential role in the maintenance of ongoing circadian rhythmicity by synchronizing SCN cells and by maintaining rhythmicity within individual neurons. To further increase the understanding of the role of VPAC2 signaling in circadian regulation, we implanted telemetric devices and simultaneously measured core body temperature, spontaneous activity, and heart rate in a strain of VPAC2-deficient mice and compared these observations with observations made from mice examined by wheel-running activity. The study demonstrates that VPAC2 signaling is necessary for a functional circadian clock driving locomotor activity, core body temperature, and heart rate rhythmicity, since VPAC2-deficient mice lose the rhythms in all three parameters when placed under constant conditions (of either light or darkness). Furthermore, although 24-h rhythms for three parameters are retained in VPAC2-deficient mice during the LD cycle, the temperature rhythm displays markedly altered time course and profile, rising earlier and peaking ～4-6 h prior to that of wild-type mice. The use of telemetric devices to measure circadian locomotor activity, temperature, and heart rate, together with the classical determination of circadian rhythms of wheel-running activity, raises questions about how representative wheel-running activity may be of other behavioral parameters, especially when animals have altered circadian phenotype.     

Placing ocular mutants into a functional context: a chronobiological approach

The behavior of mammals is characterized by a 24-h cycle of rest and activity which is a fundamental adaption to the solar cycle of light and darkness. The pacemaker of this circadian clock is localized in the ventral part of the hypothalamus, the so-called suprachiasmatic nuclei (SCN), and is entrained by light signals mediated by the eye. The eye is directly connected via the retinohypothalamic tract (RHT) to the SCN. Light that reaches the retina elicits glutamate release at the synaptic terminals of the RHT and influences the neurons in the SCN in a manner that alters the behavioral state of the animal. A light pulse that reaches the retina at the beginning of the night elicits a delay of the clock phase, whereas a light pulse that reaches the retina at the end of the dark period leads to an advance of the clock phase. This advance or delay can be quantified by measuring the change in onset of wheel-running activity. Such measurements have, and continue to provide, a remarkably powerful assay of how light is detected and transduced to regulate circadian rhythms. The methods used for such measurements in mice are described in the following article.     

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day^1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).Although circadian rhythms in running-wheel activity are typically linked to the SCN clock^6-8, circadian oscillators in many other regions of the brain and body^9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN^15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators^17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.