Cardiac Autonomic Activity During Human Sleep: Analysis of Sleep Stages and Sleep Cycles

In this study characteristics of cardiac functioning were investigated in nine subjects during their nocturnal sleep. The pre-ejection period and the high frequency component of heart rate variability were used as indices of cardiac sympathetic and parasympathetic activity of the autonomic nervous system respectively. Heart rate and the autonomic indices were assessed across physiological determined sleep stages and consecutive temporal sleep cycles. Repeated measures ANOVA analyses indicated a significant pattern of heart rate as a function of sleep stages, which was mirrored by parasympathetic activity. Further, a significant decrease of heart rate as a function of sleep cycles was mirrored by an increase of sympathetic activity. Moreover, non-REM/REM differences revealed a dominant role of parasympathetic activity during sleep stages as well as sleep cycles. These findings demonstrate that sympathetic activity is influenced by time asleep, whereas parasympathetic activity is influenced by the depth of sleep.     

Cardiac Autonomic Activity During Human Sleep: Analysis of Sleep Stages and Sleep Cycles

In this study characteristics of cardiac functioning were investigated in nine subjects during their nocturnal sleep. The pre-ejection period and the high frequency component of heart rate variability were used as indices of cardiac sympathetic and parasympathetic activity of the autonomic nervous system respectively. Heart rate and the autonomic indices were assessed across physiological determined sleep stages and consecutive temporal sleep cycles. Repeated measures ANOVA analyses indicated a significant pattern of heart rate as a function of sleep stages, which was mirrored by parasympathetic activity. Further, a significant decrease of heart rate as a function of sleep cycles was mirrored by an increase of sympathetic activity. Moreover, non-REM/REM differences revealed a dominant role of parasympathetic activity during sleep stages as well as sleep cycles. These findings demonstrate that sympathetic activity is influenced by time asleep, whereas parasympathetic activity is influenced by the depth of sleep.     

Characterization of Proteases in the Hamster Suprachiasmatic Nucleus

The activities of several proteases in the hamster suprachiasmatic nuclei were measured at different time points throughout the daily or circadian cycle. No variation for metalloproteinase A (MMP-2) activity was found, while metalloproteinase B (MMP-9) was rhythmic and maximally active during the night. In addition, diurnal variations for two low molecular weight proteases were determined, with peaks during the light phase. This rhythmicity appears to be under exogenous control, since constant darkness abolished fluctuations throughout the circadian cycle. These results suggest that protein degradation in the hamster circadian clock is regulated in a diurnal fashion.     

大鼠下丘脑交叉上核中CREB的昼夜节律

利用凝胶迁移率变化的实验方法,对饲养在光照－黑暗循环的条件和持续黑暗的条件下Wistar雄性大鼠下丘脑交叉上核中CREB含量的昼夜间变化进行了分析,发现CREB在交叉上核中具有内源性的昼夜节律.     

The Source of Transitory Innervation of Suprachiasmatic Nucleus by Tyrosine Hydroxylase-Immunoreactive Fibers during Postnatal Period in Rats

Suprachiasmatic nucleus in the rats during early postnatal development is transitorily innervated by tyrosine hydroxylase-immunoreactive fibers that are neither catecholamine- nor serotoninergic. The goal of this immunocytochemical investigation was to find out if tyrosine hydroxylase-immunoreactive neurons of anterior hypothalamus could be the source of this innervation. According to the obtained immunocytochemical data, multiple multipolar tyrosine hydroxylase-immunoreactive neurons are localized around the suprachiasmatic nucleus in the rats at days 2 and 10 of postnatal development. Most of them were observed ventrally and laterally to the nucleus. The axons of the neurons are oriented towards the suprachiasmatic nucleus. Further investigation demonstrated considerably decreased number of tyrosine hydroxylase-immunoreactive neurons surrounding the suprachiasmatic nucleus in the adult animals as compared to early postnatal period, which correlates to the number of tyrosine hydroxylase-immunoreactive fibers in this nucleus. Hence, tyrosine hydroxylase-immunoreactive neurons in the ventral region of anterior hypothalamus can be considered as a potential source of transitory innervation of suprachiasmatic nucleus by tyrosine hydroxylase-immunoreactive fibers during early postnatal development.     

Fractal Stochastic Modeling of Spiking Activity in Suprachiasmatic Nucleus Neurons

Individual neurons in the suprachiasmatic nucleus (SCN), the master biological clock in mammals, autonomously produce highly complex patterns of spikes. We have shown that most (~90%) SCN neurons exhibit truly stochastic interspike interval (ISI) patterns. The aim of this study was to understand the stochastic nature of the firing patterns in SCN neurons by analyzing the ISI sequences of 150 SCN neurons in hypothalamic slices. Fractal analysis, using the periodogram, Fano factor, and Allan factor, revealed the presence of a 1/f-type power-law (fractal) behavior in the ISI sequences. This fractal nature was persistent after the application of the GABA_A receptor antagonist bicuculline, suggesting that the fractal stochastic activity is an intrinsic property of individual SCN neurons. Based on these physiological findings, we developed a computational model for the stochastic SCN neurons to find that their stochastic spiking activity was best described by a gamma point process whose mean firing rate was modulated by a fractal binomial noise. Taken together, we suggest that SCN neurons generate temporal spiking patterns using the fractal stochastic point process.Action Editor: Carson C. Chow     

Assessment of Cardiac Autonomic Modulation during Adolescent Obesity

Objective: To investigate the cardiovascular autonomic function in pediatric obesity of different duration by using standard time domain, spectral heart rate variability (HRV), and nonlinear methods. Research Methods and Procedures: Fifty obese children (13.9 ± 1.7 years) were compared with 12 lean subjects (12.9 ± 1.6 years). Obese children were classified as recent obese (ROB) (<4 years), intermediate obese (IOB) (4 to 7 years), and long‐term obese (OB) (>7 years). In all participants, we performed blood pressure (BP) measurements, laboratory tests, and 24‐hour electrocardiogram/ambulatory BP monitoring. The spectral power was quantified in total power, very low‐frequency (LF) power, high‐frequency (HF) power, and LF to HF ratio. Total, long‐term, and short‐term time domain HRV were calculated. Poincaré plot and quadrant methods were used as nonlinear techniques. Results: All obese groups had higher casual and ambulatory BP and higher glucose, homeostasis model assessment, and triglyceride levels. All parameters reflecting parasympathetic tone (HF band, root mean square successive difference, proportion of successive normal‐to‐normal intervals, and scatterplot width) were significantly and persistently reduced in all obese groups in comparison with lean controls. LF normalized units, LF/HF, and cardiac acceleration (reflecting sympathetic activation) were significantly increased in the ROB group. In IOB and OB groups, LF, but not nonlinear, measures were similar to lean controls, suggesting biphasic behavior of sympathetic tone, whereas nonlinear analysis showed a decreasing trend with the duration of obesity. Long‐term HRV measures were significantly reduced in ROB and IOB. Discussion: Autonomic nervous system changes in adolescent obesity seem to be related to its duration. Nonlinear methods of scatterplot and quadrant analysis permit assessment of autonomic balance, despite measuring different aspects of HRV.     

Efferent Projections of Prokineticin 2 Expressing Neurons in the Mouse Suprachiasmatic Nucleus

The suprachiasmatic nucleus (SCN) in the hypothalamus is the predominant circadian clock in mammals. To function as a pacemaker, the intrinsic timing signal from the SCN must be transmitted to different brain regions. Prokineticin 2 (PK2) is one of the candidate output molecules from the SCN. In this study, we investigated the efferent projections of PK2-expressing neurons in the SCN through a transgenic reporter approach. Using a bacterial artificial chromosome (BAC) transgenic mouse line, in which the enhanced green fluorescence protein (EGFP) reporter gene expression was driven by the PK2 promoter, we were able to obtain an efferent projections map from the EGFP-expressing neurons in the SCN. Our data revealed that EGFP-expressing neurons in the SCN, hence representing some of the PK2-expressing neurons, projected to many known SCN target areas, including the ventral lateral septum, medial preoptic area, subparaventricular zone, paraventricular nucleus, dorsomedial hypothalamic nucleus, lateral hypothalamic area and paraventricular thalamic nucleus. The efferent projections of PK2-expressing neurons supported the role of PK2 as an output molecule of the SCN.     

Characterization of Melatonin-Induced FOS-Like Immunoreactivity in the Hypothalamic Suprachiasmatic Nucleus of the Rat

Abstract

The hypothalamic suprachiasmatic nucleus (SCN) is primarily responsible for the regulation of circadian rhythmicity. Melatonin, the pineal-derived neurohormone, modulates the rhythmic output of the SCN. Properly timed exposure to melatonin is able to induce changes in rhythrnic function and thereby entrain circadian rhythms of activity.

c-fos is an immediate early gene that is transiently expressed in neurons in response to receptor activation. The ventrolateral portion of the SCN (vSCN) is activated in response to phase-shifting stimuli, an event which is marked by an increase in the expression of c-fos.

In the present study, rats systemically administered the melatonin agonist 2-iodomelatonin at CT 22 demonstrated significant dose-dependent Fos immunoreactivity within the vSCN, an effect which was significantly inhibited by the melatonin antagonist N-acetyltryptamine. The Fos expression observed in the vSCN was not affected by treatment with the serotonin antagonist ketanserin or the alpha-adrenergic antagonist phentolamine. Moreover, antisense oligonucleotides to c-fos, significantly blocked the ability of 2-iodomelatonin to induce Fos expression in the vSCN at CT 22.

These results pharmacologically characterize melatonin-induced c-fos expression in the rat vSCN and provide evidence to support a c-fos-mediated mechanism through which the activation of melatonin receptors may be linked to the long-term molecular regulation of circadian rhythms controlled by the SCN.     

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca²⁺ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.     

Differential Involvement of the Suprachiasmatic Nucleus in Lipopolysaccharide-Induced Plasma Glucose and Corticosterone Responses

The hypothalamic suprachiasmatic nucleus (SCN) is an essential component of the circadian timing system, and an important determinant of neuroendocrine and metabolic regulation. Recent data indicate a modulatory role for the immune system on the circadian timing system. The authors investigated how the circadian timing system affects the hypothalamo-pituitary-adrenal (HPA) axis and glucose regulatory responses evoked by an immune challenge induced by lipopolysaccharide (LPS). LPS-induced increases in corticosterone were minimal during the trough of the daily corticosterone rhythm; in contrast, LPS effects on glucose, glucagon, and insulin did not vary across time-of-day. Complete ablation of the SCN resulted in increased corticosterone responses but did not affect LPS-induced hyperglycemia. The paraventricular nucleus (PVN) of the hypothalamus is an important neuroendocrine and autonomic output pathway for hypothalamic information, as well as one of the main target areas of the SCN. Silencing the neuronal activity in the PVN did not affect the LPS-induced corticosterone surge and only slightly delayed the LPS-induced plasma glucose and glucagon responses. Finally, surgical interruption of the neuronal connection between hypothalamus and liver did not affect the corticosterone response but slightly delayed the LPS-induced glucose response. Together, these data support the previously proposed circadian modulation of LPS-induced neuroendocrine responses, but they are at variance with the suggested major role for the hypothalamic pacemaker on the autonomic output of the hypothalamus, as reflected by the effects of LPS on glucose homeostasis. The latter effects are more likely due to direct interactions of LPS with peripheral tissues, such as the liver. (Author correspondence: a.kalsbeek@amc.uva.nl)     

The Circadian Clock Gene Period1 Connects the Molecular Clock to Neural Activity in the Suprachiasmatic Nucleus

The neural activity patterns of suprachiasmatic nucleus (SCN) neurons are dynamically regulated throughout the circadian cycle with highest levels of spontaneous action potentials during the day. These rhythms in electrical activity are critical for the function of the circadian timing system and yet the mechanisms by which the molecular clockwork drives changes in the membrane are not well understood. In this study, we sought to examine how the clock gene Period1 (Per1) regulates the electrical activity in the mouse SCN by transiently and selectively decreasing levels of PER1 through use of an antisense oligodeoxynucleotide. We found that this treatment effectively reduced SCN neural activity. Direct current injection to restore the normal membrane potential partially, but not completely, returned firing rate to normal levels. The antisense treatment also reduced baseline [Ca²⁺]i levels as measured by Fura2 imaging technique. Whole cell patch clamp recording techniques were used to examine which specific potassium currents were altered by the treatment. These recordings revealed that the large conductance [Ca²⁺]i-activated potassium currents were reduced in antisense-treated neurons and that blocking this current mimicked the effects of the anti-sense on SCN firing rate. These results indicate that the circadian clock gene Per1 alters firing rate in SCN neurons and raise the possibility that the large conductance [Ca²⁺]i-activated channel is one of the targets.     

Phosphorylation of mCRY2 at Ser557 in the Hypothalamic Suprachiasmatic Nucleus of the Mouse

Cryptochrome1 and 2 play a critical role in the molecular oscillations of the circadian clocks of central and peripheral tissues in mammals. Mouse Cryptochrome2 (mCRY2) is phosphorylated at Ser557 in the liver, in which the Ser557-phosphorylated form accumulates during the night in parallel with mCRY2 protein. Phosphorylation of mCRY2 at Ser557 allows subsequent phosphorylation at Ser553 by glycogen synthase kinase-3β (GSK-3β), resulting in efficient degradation of mCRY2 by a proteasome pathway. In the present study, we found that mCRY2 is phosphorylated at Ser557 also in the region of the mouse brain containing the suprachiasmatic nucleus (SCN), the central circadian clock tissue. Daily fluctuation of the Ser557-phosphorylation level in the SCN region suggests an important role of sequential phosphorylation of Ser557 and Ser553 in the rhythmic degradation of mCRY2 in both central and peripheral clocks of mice.