大鼠交叉上核中SS和VIPmRNA昼夜节律的研究

用Ｎｏｒｔｈｅｒｎｂｌｏｔ杂交方法分析ＬＤ循环条件下大鼠ＳＣＮ和ＣＸ的ＳＳｍＲＮＡ和ＶＩＰｍＲＮＡ丰度的昼夜变化，结果表明这两种ｍＲＮＡ昼夜间的相对含量在ＣＸ中基本不变，而在ＳＣＮ中则呈现规律性变化的模式，与双侧眼球摘除后大鼠ＳＣＮｍＲＮＡ丰度昼夜变化的实验结果相比较，ＳＳｍＲＮＡ丰度变化不受外界光的影响，具有内源性的昼夜节律，而ＶＩＰｍＲＮＡ丰度的昼夜变化则受外界光的影响。     

哺乳动物昼夜节律组构中的下丘脑视交叉上核和松果腺

哺乳动物下丘脑视交叉上核（SCN）是昼夜节律最主要的起搏器,控制着机体的生理和行为的节律。它具有自身内在的节律性,同时也受光照周期信号和一些内源性化学物质的调节。检查腺分泌裉黑素（MEL）受SCN的调控,MEL通过作用于SCN上高亲和性MEL受体,启动第二、第三信使系统,调整SCN的昼夜节律活动。这种调整具有时间敏感性。     

树Qu尿中皮质醇浓度的昼夜节律及其在视交叉上核损毁后的变化

用放射免疫方法对连续采集的正常树Ｑｕ及视交叉上核损毁树Ｑｕ尿中的皮质醇进行了测定,以分析其尿中皮质醇浓度及排泄量的昼夜变化规律,结果表明,正常树Ｑｕ尿中皮质醇的浓度及单位时间内在排泄均具有明显的昼夜节律,在一天中,１１：００前后的浓度最高,０：００前后的浓度最低,前者约为８倍,视交叉上核损毁后,树Ｑｕ皮质醇的这种昼夜节律消失,说明视交叉上核在树Ｑｕ中也是机体昼夜节律重要的振荡器。     

树Qu排尿的昼夜节律及其在视交叉上核损毁后的变化

显探讨树Ｑｕ视交叉上核是否参与泌尿昼夜节律的调控,对正常树Ｑｕ及视交叉上核损毁后的树Ｑｕ排尿的昼夜时间规律进行了观测分析。结果表明,正常树Ｑｕ排尿呈现明显的昼夜节律－－白天尿量为全天尿量的（８８．５２±１５．０４）％;视交叉上核损毁后,树Ｑｕ排尿的昼夜节律明显改变－白天尿量仅为全天尿量的（６２．８６±１８．１８）％,同时树Ｑｕ的尿量（特别是夜间的尿量）明显增多。以上结果说明视交叉上核在树Ｑｕ体内也     

褪黑素对大鼠下丘脑薄片视交叉上核神经元放电昼夜节律性的调制

先用持续光照和松果腺切除预处理大鼠,然后制成下丘脑薄片,记录其视交叉上核（ＳＣＮ）神经元自发放电,观察其昼夜变化和褪黑素（ＭＥＬ）对它的影响。实验结果表明：⑴在正常光照（光照：黑暗＝１２：１２）条件下,ＳＣＮ神经元自发放电频率呈现昼夜低的节律性。在昼夜时间（ＣＴ）６－８出现放电高峰,频率约为８．３Ｈｚ;在ＣＴ１８－２０出现低谷,频率约为３．８Ｈｚ。松果腺切除后,ＳＣＮ神经元自发放电的昼夜节律性基本     

大鼠视交叉上核与松果体中Clock基因转录的昼夜节律性及不同光反应性

本研究旨在观察和比较视交叉上核（suprachiasmatic nucleus,SCN）与松果体（pineal gland,pG）中Clock基因内源性昼夜转录变化规律以及光照对其的影响。Sprague-Dawley大鼠在持续黑暗（constant darkness,DD）和12h光照：12h黑暗交替（12hourlight：12hour-darkcycle,LD）光制下分别被饲养8周（n=36）和4周n=36）后,在一昼夜内每隔4h采集一组SCN和PG组织（n=6）,提取总RNA,用竞争性定量RT-PCR测定不同昼夜时点（circadian times．CT or zeitgeber times．ZT）各样品中Clock基因的mRNA相对表达量,通过余弦法和ClockLab软件获取节律参数,并经振幅检验是否存在昼夜节律性转录变化。结果如下：（1）SCN中Clock基因mRNA的转录在DD光制下呈现昼低夜高节律性振荡变化（P〈0．05）,PG中Clock基因的转录也显示相似的内源性节律外观,即峰值出现于主观夜晚（SCN为CTl5,PG为CT18）,谷值位于主观白天（SCN为CT3,PG为CT6）（P〉0．05）。（2）LD光制下SCN中Clock基因的转录也具有昼夜节律性振荡（P〈0．05）,但与其DD光制下节律外观相比,呈现反时相节律变化（P〈0．05）,且其表达的振幅及峰值的mRNA水平均增加（P〈0．05）,而PG中Clock基因在LD光制下转录的相应节律参数变化却恰恰相反（P〈0．05）。（3）在LD光制下,光照使PG中Clock基因转录的节律外观反时相于SCN（P〈0．05）,即在SCN和PG的峰值分别出现于光照期ZT10和黑暗期ZT17,谷值分别位于黑暗期ZT22和光照期ZT5。结果表明,Clock基因的昼夜转录在SCN和PG中存在同步的内源性节律本质,而光导引在这两个中枢核团调节Clock基因昼夜节律性转录方面有着不同的作用。     

视交叉上核的内源性昼夜节律以及光,谷氨酸和NO的调制作用

自从发现视交叉上核（ＳＣＮ）中有直接的视网膜下丘脑投射纤维以来,ＳＣＮ的内源性节律及其调节机制受到广泛重视,已成为令人感兴趣的新课题。哺乳动物的２４ｈ昼夜节律而言,ＳＣＮ是主要的启步者,但ＳＣＮ内源性的振荡节律又受到环境光暗周期、谷氨酸和一氧化氮的拖拽。     

树■排尿的昼夜节律及其在视交叉上核损毁后的变化

为探讨树视交叉上核是否参与泌尿昼夜节律的调控,对正常树及视交叉上核损毁后的树排尿的昼夜时间规律进行了观测分析。结果表明,正常树排尿呈现明显的昼夜节律———白天尿量为全天尿量的（８８５２±１５４０）％;视交叉上核损毁后,树排尿的昼夜节律明显改变———白天尿量仅为全天尿量的（６２８６±１８１８）％,同时树的尿量（特别是夜间的尿量）明显憎多。以上结果说明视交叉上核在树体内也参与了泌尿昼夜节律的调节。     

树尿中皮质醇浓度的昼夜节律及其在视交叉上核损毁后的变化

用放射免疫方法对连续采集的正常树及视交叉上核损毁树尿中的皮质醇进行了测定,以分析其尿中皮质醇浓度及排泄量的昼夜变动规律。结果表明,正常树尿中皮质醇的浓度及单位时间内的排泄量均具有明显的昼夜节律,在一天中,１１００前后的浓度最高,０００前后的浓度最低,前者约为后者的８倍。视交叉上核损毁后,树皮质醇的这种昼夜节律消失,说明视交叉上核在树中也是机体昼夜节律重要的振荡器。     

眼镜蛇毒对大鼠下丘脑视上核NOS表达的影响

目的观察眼镜蛇毒对大鼠视上核NOS表达的影响。方法将SD大鼠随机分为正常对照组、生理盐水组和眼镜蛇毒组,采用免疫组织化学染色法,观察并比较NOS阳性神经元在各组大鼠下丘脑视上核的分布情况。结果眼镜蛇毒组大鼠视上核NOS阳性神经元比生理盐水组、正常对照组表达明显增强(P0.01)。结论眼镜蛇毒对大鼠下丘脑视上核NOS的表达有上调作用。     

Perturbing Circadian Oscillations in an In Vitro Suprachiasmatic Nucleus With Magnetic Stimulation

Many neurological disorders are associated with abnormal oscillatory dynamics. The suprachiasmatic nucleus (SCN) is responsible for the timing and synchronization of physiological processes. We performed experiments on PERIOD2::LUCIFERASE transgenic “knock-in” mice. In these mice, a gene that is expressed in a circadian pattern is fused to an inserted gene that codes for luciferase, which is a bioluminescent enzyme. A one-time 3 min magnetic stimulation (MS) was applied to excised slices of the SCN. The MS consisted of a 50-mT field that was turned on and off 4,500 times. The rise time and fall time of the field were 75 μs. A photon count that extended over the full 5 days that the slice remained viable, subsequently revealed how the MS affected the circadian cycle. The MS was applied at points in the circadian cycle that correspond to either maximal or minimal bioluminescence. It was found that both the amplitude and period of the endogenous circadian oscillation are affected by MS and that the effects strongly depend on where in the circadian cycle the stimulation was applied. Our MS dose is in the same range as clinically applied doses, and our findings imply that transcranial MS may be instrumental in remedying disorders that originate in circadian rhythm abnormalities. Bioelectromagnetics. 2020;41:63–72 © 2019 Wiley Periodicals, Inc.     

Effect of Continuous Infusion of Anti-L1 Antibody into the Third Cerebral Ventricle above the Suprachiasmatic Nucleus on the Circadian Rhythm of Locomotor Activity in Rats

During an investigation into the role of the neural cell adhesion molecules such as L1 and NCAM in the generation mechanism of circadian rhythms, we observed that L1-like immunoreactive substance is expressed in the hypothalamic suprachiasmatic nucleus (SCN). Therefore, we examined the effect of continuous infusion of anti-L1 antibody into the third cerebral ventricle above the SCN using an Alzet osmotic minipump, on the circadian rhythm of locomotor activity in rats under constant red dim light (less than 1 lx) condition, in order to elucidate the role of L1 in the mechanism of circadian rhythm. Continuous infusion of intact rabbit IgG into the third cerebral ventricle above the SCN, which was done as a control experiment, shifted the phase of the free-running circadian rhythm and reduced daily locomotor activity for an initial few days, however, it did not eliminate the circadian rhythm. In contrast, continuous infusion of anti-L1 antibody temporarily disrupted the circadian rhythm during the infusion period. Furthermore, the infusion of the anti-L1 antibody but not that of control IgG caused a change in the SCN conformation, from which it appeared that SCN neurons displaced in dorsal direction, 4 days after the start of the infusion. These findings suggest that the cell adhesion molecule, L1, might be involved in the generation and/or transduction of the time signal of the circadian rhythm in the SCN.     

Circadian Rhythm in Muscarinic Receptor Subtypes in Rat Forebrain

The binding of different ligands to muscarinic receptors in the centra) nervous system is regulated by several factors. Among these are the administration of drugs, disease, ontogeny or aging. Studies carried out in rat brains have demonstrated changes in the density of the muscarinic receptors at different times of the day. These changes might be related to variations in the circadian rhythms. In this work we have studied the binding of the [3H]-N-methyl-escopolamine, the agonist carbachol and the antagonist pirenzepine to muscarinic receptors in rat forebrains at 10.00,14.00,18.00,22.00,02.00 and 06.00 hr. We have observed changes in the density of muscarinic receptors but not changes in affinity to the radioligand. The Bmax values obtained by saturation studies were maximum at 14.00 hr and minimum at 02.00 hr (p < 0.05 Mann-Whitney's test). Inhibition studies in the presence of the non-selective agonist carbachol and the selective antagonist pirenzepine, at the same time-points, did not show statistically significant changes in the Bmax values. These data indicate that changes in the Bmax values are only observed in the total population of muscarinic receptors and are not due to modifications in the subtypes of muscarinic receptors nor to the different affinity states of agonist binding.     

Reduced Neurophysin Immunoreactivity in Rat Suprachiasmatic Nucleus Parallels Dissociation of Circadian Feeding Rhythm in Constant Light

Several distinct neuronal populations can be outlined in the suprachiasmatic nucleus (SCN) by employing immunohistochemistry. Understanding their interaction may serve as the key to the processes involved in the generation of circadian rhythms by the SCN. 15 adult rats were exposed to constant dim light (LL) and 3 animals as controls to an LD 12:12 light schedule over 140 days. When sacrificed 10 of the LL-animals had lost their circadian feeding rhythm while 5 were free-running and the controls kept an entrained rhythm. The brains were immunohistochemically stained for myelin basic protein, neurophysin (NPH), vasoactive intestinal peptide, neuropeptide Y, synaptophysin and the leucocyte epitopes FAL and HNK-1. Demarcation of intensely and very intensely stained NPH-positive areas by subjective gray-level-discrimination and computerized area measurement revealed that in rhythmic rats (n=8) the areas containing the stained material were twice as large (0.06 ± 0.03 mm2 vs. 0.028 ± 0.027 mm2; p=0.05) than in arrhythmic animals. It is hypothesized that low NPH-contents in arrhythmic animals reflect arrest of the ‘clockwork’ in the SCN at circadian time 12:00.     

Reduced Neurophysin Immunoreactivity in Rat Suprachiasmatic Nucleus Parallels Dissociation of Circadian Feeding Rhythm in Constant Light

Several distinct neuronal populations can be outlined in the suprachiasmatic nucleus (SCN) by employing immunohistochemistry. Understanding their interaction may serve as the key to the processes involved in the generation of circadian rhythms by the SCN. 15 adult rats were exposed to constant dim light (LL) and 3 animals as controls to an LD 12:12 light schedule over 140 days. When sacrificed 10 of the LL-animals had lost their circadian feeding rhythm while 5 were free-running and the controls kept an entrained rhythm. The brains were immunohistochemically stained for myelin basic protein, neurophysin (NPH), vasoactive intestinal peptide, neuropeptide Y, synaptophysin and the leucocyte epitopes FAL and HNK-1. Demarcation of intensely and very intensely stained NPH-positive areas by subjective gray-level-discrimination and computerized area measurement revealed that in rhythmic rats (n=8) the areas containing the stained material were twice as large (0.06 ± 0.03 mm2 vs. 0.028 ± 0.027 mm2; p=0.05) than in arrhythmic animals. It is hypothesized that low NPH-contents in arrhythmic animals reflect arrest of the 'clockwork' in the SCN at circadian time 12:00.     

Dynamical Heterogeneity of Suprachiasmatic Nucleus Neurons Based on Regularity and Determinism

The suprachiasmatic nucleus (SCN) is known to be the master biological clock in mammals. Despite the periodic mean firing rate, interspike interval (ISI) patterns of SCN neurons are quite complex and irregular. The aim of the present study was to investigate the existence of nonlinear determinism in the complex ISI patterns of SCN neurons. ISI sequences were recorded from 173 neurons in rat hypothalamic slice preparations using a cell-attached patch recording technique. Their correlation dimensions (D₂) were estimated, and were then compared with those of the randomly-shuffled surrogate data. We found that only 16 neurons (16/173) exhibited deterministic ISI patterns of spikes. In addition, clustering analysis revealed that SCN neurons could be divided into two subgroups of neurons each having distinct values of coefficient of variation (CV) and skewness (SK). Interestingly, most deterministic SCN neurons (14/16) belonged to the group of irregularly spiking neurons having large CV and SK values. To see if the neuronal coupling mediated by the γ-aminobutyric acid (GABA), the major neurotransmitter in the SCN, contributed to the deterministic nature, we examined the effect of the GABA_A receptor antagonist bicuculline on D₂ values of 56 SCN neurons. 8 SCN neurons which were originally stochastic became to exhibit deterministic characteristics after the bicuculline application. This result suggests that the deterministic nature of the SCN neurons arises not from GABAergic synaptic interactions, but likely from properties inherent to neurons themselves.Action Editor: Barry J. Richmond     

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     

Circadian Rhythm and Seasonal Variations in Basal cAMP Content of Rat Heart Ventricles

The cAMP content in rat heart ventricles was studied at 3-hr intervals during 24hr at different times of the year. A significant circadian rhythm in cAMP content was found. Time of the year reproducibly influenced the 24-hr mean, the amplitude as well as the peak value in cAMP in relation to circadian time.     

Circadian Activity Rhythms and Phase-Shifting of Cultured Neurons of the Rat Suprachiasmatic Nucleus

The mammalian suprachiasmatic nucleus (SCN) is the major endogenous pacemaker that coordinates various daily rhythms including locomotor activity and autonomous and endocrine responses, through a neuronal and humoral influence. In the present study we examined the behavior of dispersed individual SCN neurons obtained from 1- to 3-day-old rats cultured on multi-microelectrode arrays (MEAs). SCN neurons were identified by immunolabeling for the neuropeptides arginine-vasopressin (AVP) and vasoactive intestinal polypeptide (VIP). Single SCN neurons cultured at low density onto an MEA can express firing rate patterns with different circadian phases. In these cultures we observed rarely synchronized firing patterns on adjacent electrodes. This suggests that, in cultures of low cell densities, SCN neurons function as independent pacemakers. To investigate whether individual pacemakers can be influenced independently by phase-shifting stimuli, we applied melatonin (10 pM to 100 nM) for 30 min at different circadian phases and continuously monitored the firing rate rhythms. Melatonin could elicit phase-shifting responses in individual clock cells which had no measurable input from other neurons. In several neurons, phase-shifts occurred with a long delay in the second or third cycle after melatonin treatment, but not in the first cycle. Phase-shifts of isolated SCN neurons were also observed at times when the SCN showed no sensitivity to these phase-shifting stimuli in recordings from brain slices. This finding suggests that the neuronal network plays an essential role in the control of phase-shifts.