Circadian rhythms in gastrin receptors in rat fundic stomach

Circadian rhythmicity in the number of gastrin receptors in rat fundic mucosa was characterized and was related to the concentrations of gastrin in serum and in antrum. Male Sprague-Dawley rats were acclimated to 12 hr light alternating with 12 hr darkness. Subgroups of six rats each were killed at 4-hr intervals. Fundic mucosa was collected for measurement of gastrin receptors; serum and antral tissues were collected for measurement of gastrin levels by radioimmunoassay. Circadian periodicity in the data was determined by cosinor analyses. In both freely fed and fasted rats, gastrin receptors showed circadian variation (range 2.5–10 fmol/mg protein), as did serum gastrin concentrations (range in fed rats 195–407 pg/ml). The phasing of the intrinsic circadian variation in gastrin receptor level that was observed in the fasted rats was advanced by a few hours in fed rats. This shift is probably due to food-induced gastrin release, resulting in gastrin-mediated down-regulation of gastrin receptors, followed by up-regulation of gastrin receptors. Food-related effects were thus superimposed upon the intrinsic circadian rhythms in gastrin receptor levels, causing the circadian variation in gastrin receptor levels in the fed rats to be shifted forward compared to that in the fasted rats. No significant circadian rhythms, on the other other hand, were found in concentrations of gastrin in the antrum. These results suggest that changes in sensitivity of target tissues to hormones are related to both intrinsic circadian rhythms in levels of hormone receptors and also to food-related changes in hormone-receptor levels mediated by changing serum hormone levels.Supported by grants from the National Institutes of Health (RO1 DK 15421, PO1 DK 35608, CA 38651).     

Circadian rhythms in the aged: a review

After a review of the fundamental concepts on chronobiology, the importance of circadian rhythms in the aged was examined on the basis of the data obtained in animals and humans, including personal observations on over 40 blood constituents. During ageing there are significant modifications of circadian rhythms, with frequent diminution of amplitude and a shift of acrophase. The biological, clinical and therapeutic implications of these findings are discussed.     

Circadian rhythms of plasma melatonin in the ruin lizard Podarcis sicula: Effects of pinealectomy

Plasma melatonin was measured in lizards (Podarcis sicula) at six different times of day under conditions of constant temperature and darkness. Intact animals showed a circadian rhythm of melatonin with a peak in the subjective night of 207 pg/ml (median) and a trough during the subjective day that was below the minimum detection level of the assay (50 pg/ml). Pinealectomy abolished the circadian rhythm of plasma melatonin; median levels were near or below the minimum detection level at all times sampled. The data suggest that the pineal is the only source of rhythmic blood-borne melatonin in Podarcis sicula, and are consistent with the hypothesis that changes in the free-running period of the locomotor rhythm induced by pinealectomy in this species are due to withdrawal of rhythmic melatonin from the blood.     

CaV3.1 is a tremor rhythm pacemaker in the inferior olive

The rhythmic motor pathway activation by pacemaker neurons or circuits in the brain has been proposed as the mechanism for the timing of motor coordination, and the abnormal potentiation of this mechanism may lead to a pathological tremor. Here, we show that the potentiation of Ca_V3.1 T-type Ca²⁺ channels in the inferior olive contributes to the onset of the tremor in a pharmacological model of essential tremor. After administration of harmaline, 4- to 10-Hz synchronous neuronal activities arose from the IO and then propagated to cerebellar motor circuits in wild-type mice, but those rhythmic activities were absent in mice lacking Ca_V3.1 gene. Intracellular recordings in brain-stem slices revealed that the Ca_V3.1-deficient inferior olive neurons lacked the subthreshold oscillation of membrane potentials and failed to trigger 4- to 10-Hz rhythmic burst discharges in the presence of harmaline. In addition, the selective knockdown of Ca_V3.1 gene in the inferior olive by shRNA efficiently suppressed the harmaline-induced tremor in wild-type mice. A mathematical model constructed based on data obtained from patch-clamping experiments indicated that harmaline could efficiently potentiate Ca_V3.1 channels by changing voltage-dependent responsiveness in the hyperpolarizing direction. Thus, Ca_V3.1 is a molecular pacemaker substrate for intrinsic neuronal oscillations of inferior olive neurons, and the potentiation of this mechanism can be considered as a pathological cause of essential tremor.     

Hugin+ neurons provide a link between sleep homeostat and circadian clock neurons

Sleep is controlled by homeostatic mechanisms, which drive sleep after wakefulness, and a circadian clock, which confers the 24-h rhythm of sleep. These processes interact with each other to control the timing of sleep in a daily cycle as well as following sleep deprivation. However, the mechanisms by which they interact are poorly understood. We show here that hugin⁺ neurons, previously identified as neurons that function downstream of the clock to regulate rhythms of locomotor activity, are also targets of the sleep homeostat. Sleep deprivation decreases activity of hugin⁺ neurons, likely to suppress circadian-driven activity during recovery sleep, and ablation of hugin⁺ neurons promotes sleep increases generated by activation of the homeostatic sleep locus, the dorsal fan-shaped body (dFB). Also, mutations in peptides produced by the hugin⁺ locus increase recovery sleep following deprivation. Transsynaptic mapping reveals that hugin⁺ neurons feed back onto central clock neurons, which also show decreased activity upon sleep loss, in a Hugin peptide–dependent fashion. We propose that hugin⁺ neurons integrate circadian and sleep signals to modulate circadian circuitry and regulate the timing of sleep.

Sleep is regulated by two processes, circadian and homeostatic (1). The endogenous circadian clock, together with its downstream pathways, is synchronized to external day–night cycles and determines the timing of sleep to produce 24-h rhythms in sleep and wake. The homeostatic process tracks sleep:wake history and generates sleep drive, based on the extent of wakefulness. Overtly, sleep homeostasis can be seen as an increase in sleep duration and depth after prolonged wakefulness. Generally, circadian and homeostatic processes are studied as separate mechanisms that regulate sleep, but they clearly intersect and are coordinated in a daily cycle to promote the onset and maintenance of sleep at night. Following sleep deprivation, the homeostatic system can drive sleep at the wrong time of day, but even under these conditions, interactions between the two systems determine the timing and duration of sleep. However, the neuronal mechanisms by which circadian and homeostatic pathways signal to each other are largely unknown.The functions and regulation of sleep are extensively studied in model organisms, such as Drosophila melanogaster (2). In the Drosophila brain, the circadian clock is expressed in ∼150 clock neurons that are organized into neuroanatomical groups, of which the ventral and dorsal lateral neurons are the most important for driving rhythms of locomotor activity (3, 4). The small ventrolateral neurons (s-LNvs) link to other brain regions through different circuits. One of those circuits connects the s-LNvs to the site of the motor ganglion, the thoracic nerve cord: s-LNvs → DN1s → Dh44⁺ neurons → hugin⁺ neurons→ ventral nerve cord (5). Dh44-expressing neurons in the pars intercerebralis regulate locomotor activity rhythms in part through the signaling of DH44 neuropeptide to hugin-expressing neurons in the subesophageal zone (SEZ) (6, 7). Dh44⁺ and hugin⁺ circadian output neurons do not contain canonical molecular clocks, but display cycling in neuronal activity or peptide release, likely under control of upstream circadian signals (7–9). Links between these neurons and loci regulating sleep homeostasis have not been identified yet.Regulation of sleep homeostasis in flies involves the central complex and mushroom body (10–13). Of particular importance is a group of sleep-promoting neurons labeled by the 23E10-GAL4 driver that projects to the dorsal fan-shaped body (dFB) neuropil in the central complex. These dFB neurons promote sleep when activated (14, 15), and they are required for normal sleep rebound after deprivation (16). 23E10⁺ neurons receive input signals from R5 ellipsoid body neurons, which track sleep need (17).As hugin⁺ neurons are significantly downstream of central clock neurons and close to behavioral outputs, we asked whether they also have a role in sleep. We find that hugin⁺ neurons are dispensable for determining daily sleep amount, but they show decreases in activity following sleep deprivation. They also receive projections from the dFB and counter sleep-promoting effects of 23E10⁺ neurons, such that ablation of hugin⁺ neurons enhances sleep driven by 23E10⁺ cells. Further supporting a role in sleep, mutations in Hugin peptides affect recovery sleep after deprivation and enhance sleep driven by 23E10⁺ cells. hugin⁺ neurons target PDF⁺ s-LNv clock neurons, which also show decreases in intracellular Ca²⁺ levels following sleep deprivation. Thus hugin⁺ neurons serve as an integrations site of signals from both the sleep homeostat and the circadian clock.     

Daily serotonin rhythms in rat brain during postnatal development and aging

Aging is characterized by progressive decline in most physiological functions. The age-related sleep disturbances have been attributed to disturbances of circadian function. Neurotransmitter serotonin plays important role in the photic and non-photic regulation of circadian rhythms and is a precursor of melatonin, an internal zeitgeber. To understand the age induced changes in the functional integrity of circadian system, we studied daily serotonin rhythms in brain by measuring serotonin levels at variable time points in wide range of age groups such as 15 days, 1, 2, 3 (adult), 4, 6, 9, 12, 18 and 24-months old male Wistar rats. Animals were maintained under light-dark conditions (LD 12:12), 2 weeks prior to experiment. We report here, mean serotonin levels over 24 h period in brain is highest at 3 months and daily serotonin rhythmicity reliably begins at 3 months and disintegrates at middle age and beyond. The age induced changes in daily serotonin rhythmicity in brain obtained in present study will be a step towards understanding age induced disorders of circadian function.     

Melatonin regulates circadian electroretinogram rhythms in a dose- and time-dependent fashion

The circadian rhythm of the chick electroretinogram (ERG) is regulated by the indoleamine hormone melatonin. To determine if the concentration of melatonin or the time at which it was administered would have differential effects on ERG parameters, we conducted experiments analyzing the effects of melatonin at different times of the day. Circadian rhythms of a- and b-wave implicit times and amplitudes were observed in both light:dark (LD) and in continuous darkness (DD). Intramuscular melatonin administration of 1 mg/kg and 100 ng/kg decreased a- and b-wave amplitudes and increased a- and b-wave implicit times. This effect was significantly greater than that observed for 1 ng/kg melatonin, which had little to no effect over the saline controls. The effect of 1 mg/kg and 100 ng/kg melatonin on a- and b-wave amplitude in LD and on b-wave amplitude in DD was greater during the night (ZT/CT 17) than during the day (ZT/CT 5). The fold change in b-wave implicit time over that of controls was greater during the day (ZT/CT 5) than during the night (ZT/CT 17). These data indicate that melatonin may play a role in regulating a day and night functional shift in the retina, and that it does so via regulation of a retinal clock.     

Melatonin Rhythms in Quail: Regulation by Photoperiod and Circadian Pacemakers

The profile of melatonin in the eyes, pineal, and blood of Japanese quail was assessed in birds held under LD 16:8 and LD 6: 18 photoperiods. Melatonin levels in all three tissues showed a robust daily rhythm with higher levels occurring at night. The amplitude of the rhythm was depressed and its duration lengthened on LD 6: 18 relative to LD 16:8. The blood melatonin rhythm precisely reflected the rhythms shown by the pineal and eyes, supporting the idea that the blood rhythm is a result of melatonin secretion by both the eyes and pineal.
The ocular melatonin rhythm continued after sectioning of the optic nerve, was reentrainable to a shift in the phase of the LD cycle, and persisted for at least 2 days in constant darkness. It was concluded that either (1) an intraocular circadian clock drives the ocular melatonin rhythm, or (2) an extraocular clock drives the ocular melatonin rhythm via a route other than the efferent innervation (which enters the eye via the optic tract).     

巨噬细胞移动抑制因子对HL-1细胞T型钙电流的调控

目的 观察巨噬细胞移动抑制因子（macrophage migration inhibitory factor,MIF）对心房肌细胞T型钙电流（T-type calcium channel current,ICa,T）的调控.方法 使用全细胞膜片钳和分子生物分析方法检测心房肌细胞ICa,T的表达.结果 在体外培养的心房肌细胞株（HL-1细胞）中,小鼠重组MIF（20、40 nmol/L,24 h）可明显抑制ICa,T的峰值电流,与对照组比较,差异均有统计学意义[峰值内向电流：（-17.5±2.9）pA/pF vs.（-27.9±3.4） pA/pF,P＜0.05;（-11.3±1.7）pA/pF vs.（-27.9±3.4）pA/pF,P＜0.01];并可损伤电压依赖的ICa,T激活,使T型钙通道α1G和α1H亚单位mRNA表达下调.而Src非特异性抑制剂genistein和特异性抑制剂PP1可逆转40 nmol/L MIF所致的ICa,T下调[genistein：（-11.3±1.7）pA/pF vs.（-16.1±0.8）,P＜0.05;PPI：（-11.3±1.7）pA/pFvs.（-19.0±3.2）pA/pF,P＜0.05].结论 MIF可能通过影响ICa,T参与心房颤动的病理过程,Src可能参与该信号转导途径.     

Early chronotype with advanced activity rhythms and dim light melatonin onset in a rural population

Studying communities at different stages of urbanisation and industrialisation can teach us how timing and intensity of light affect the circadian clock under real-life conditions. We have previously described a strong tendency towards morningness in the Baependi Heart Study, located in a small rural town in Brazil. Here, we tested the hypothesis that this morningness tendency is associated with early circadian phase based on objective measurements (as determined by dim light melatonin onset, DLMO, and activity) and light exposure. We also analysed how well the previously collected chronotype questionnaire data were able to predict these DLMO values. The average DLMO observed in 73 participants (40 female) was 20:03 ± 01:21, SD, with an earlier average onset in men (19:38 ± 01:16) than in women (20:24 ± 01:21; P ≤ .01). However, men presented larger phase angle between DLMO and sleep onset time as measured by actigraphy (4.11 hours vs 3.16 hours; P ≤ .01). Correlational analysis indicated associations between light exposure, activity rhythms and DLMO, such that early DLMO was observed in participants with higher exposure to light, higher activity and earlier light exposure. The strongest significant predictor of DLMO was morningness-eveningness questionnaire (MEQ) (beta=−0.35, P ≤ .05), followed by age (beta = −0.47, P ≤ .01). Sex, light exposure and variables derived from the Munich chronotype questionnaire were not significant predictors. Our observations demonstrate that both early sleep patterns and earlier circadian phase have been retained in this small rural town in spite of availability of electrification, in contrast to metropolitan postindustrial areas.     

Effect of light treatment on sleep and circadian rhythms in demented nursing home patients

OBJECTIVES: To determine whether fragmented sleep in nursing home patients would improve with increased exposure to bright light. DESIGN: Randomized controlled trial. SETTING: Two San Diego-area nursing homes. PARTICIPANTS: Seventy-seven (58 women, 19 men) nursing home residents participated. Mean age +/- standard deviation was 85.7 +/- 7.3 (range 60-100) and mean Mini-Mental State Examination was 12.8 +/- 8.8 (range 0-30). INTERVENTIONS: Participants were assigned to one of four treatments: evening bright light, morning bright light, daytime sleep restriction, or evening dim red light. MEASUREMENTS: Improvement in nighttime sleep quality, daytime alertness, and circadian activity rhythm parameters. RESULTS: There were no improvements in nighttime sleep or daytime alertness in any of the treatment groups. Morning bright light delayed the peak of the activity rhythm (acrophase) and increased the mean activity level (mesor). In addition, subjects in the morning bright light group had improved activity rhythmicity during the 10 days of treatment. CONCLUSION: Increasing exposure to morning bright light delayed the acrophase of the activity rhythm and made the circadian rhythm more robust. These changes have the potential to be clinically beneficial because it may be easier to provide nursing care to patients whose circadian activity patterns are more socially acceptable.     

Diabetes-induced changes in calcium homeostasis and the effects of calcium channel blockers in rat and mice nociceptive neurons

Aims/hypothesis: Distal neuropathy is the most common complication of diabetes mellitus, making it important to reveal the cellular mechanisms leading to its development, one of which might be the alteration in intracellular calcium homeostasis in primary and secondary nociceptive neurons. We aimed to investigate these possible changes. Methods: Control and streptozotocin-treated diabetic rats and mice were used. Changes in intracellular free calcium concentrations ([Ca²⁺]_i) were measured fluorometrically in primary nociceptive neurons from dorsal root ganglia and in secondary nociceptive neurons from substantia gelatinosa of spinal dorsal horn slices. Results: Measurements of [Ca²⁺]_i increases induced in dorsal root ganglion and dorsal horn neurons by membrane depolarization did not show any substantial difference in their peak amplitudes in control and diabetic animals. However, a definite prolongation of the decay phase of the transients was observed under diabetic conditions. Caffeine application to dorsal root ganglion and dorsal horn neurons induced a transient elevation of [Ca²⁺]_i which was less prominent in cells from diabetic animals. Short-term application of a calcium channel blocker nifedipine showed a substantial amplification of its action in diabetic neurons. However, chronic administration of nimodipine induced a clear increase in the peak values of transients in dorsal root ganglion neurons of diabetic animals compared with those of untreated animals. Conclusion/interpretation: The described changes of calcium signalling in nociceptive neurons could be the reason for the development of distal polyneuropathy and its symptoms in the early stages of diabetes mellitus. [Diabetologia (2001) 44: 1302–1309] Received: 28 December 2000 and in revised form: 25 May 2001     

Circadian rhythms of gastric mucus efflux and residual mucus gel in the fasting rat stomach

We hypothesized that two putative gastric protective factors, mucus efflux and residual mucus gel content, would manifest circadian rhythms, as reported in several other gastric functions. Rats were adapted for three weeks on a 12-hr light schedule, fasted 18-hr and studied at 3-hr intervals. Under anesthesia, the stomachs were cannulated and filled with test solution. Thirty minutes later, they were drained and the luminal fluid was analyzed for mucus content by Alcian blue binding. Residual mucus gel was determined by direct injection of dye into the lumen. Alcian blue binding of rat mucus was expressed as equivalent milligrams of porcine mucin. Both parameters showed a significant (P<0.001) circadian rhythm. Mucus efflux peaked at 5:03±0:52 HALO (hours after lights on), and residual mucus at 6:00±0.46 HALO. Thus, the interplay of circadian rhythms in aggressive and defensive gastric mucosal functions is supported.     

Calcium signaling and T-type calcium channels in cancer cell cycling

Regulation of intracellular calcium is an important signaling mechanism for cell proliferation in both normal and cancerous cells. In normal epithelial cells, free calcium concentration is essential for cells to enter and accomplish the S phase and the M phase of the cell cycle. In contrast, cancerous cells can pass these phases of the cell cycle with much lower cytoplasmic free calcium concentrations, indicating an alternative mechanism has developed for fulfilling the intracellular calcium requirement for an increased rate of DNA synthesis and mitosis of fast replicating cancerous cells. The detailed mechanism underlying the altered calcium loading pathway remains unclear; however, there is a growing body of evidence that suggests the T-type Ca^2＋ channel is abnormally expressed in cancerous cells and that blockade of these channels may reduce cell proliferation in addition to inducing apoptosis. Recent studies also show that the expression of T-type Ca^2＋ channels in breast cancer cells is proliferation state dependent, i.e. the channels are expressed at higher levels during the fast-replication period, and once the cells are in a non-proliferation state, expression of this channel is minimal. Therefore, selectively blocking calcium entry into cancerous cells may be a valuable approach for preventing tumor growth. Since T-type Ca^2＋ channels are not expressed in epithelial cells, selective T-type Ca^2＋ channel blockers may be useful in the treatment of certain types of cancers.     

Classification of circadian pain rhythms and pain characteristics in chronic pain patients: An observational study

This study aimed to perform cluster analysis in patients with chronic pain to extract groups with similar circadian rhythms and compare neuropathic pain and psychological factors among these groups to identify differences in pain-related outcomes. A total of 63 community-dwellers with pain lasting at least 3 months and Numerical Rating Scale scores of ≥2 were recruited from 3 medical institutions. Their pain circadian rhythms were evaluated over 7 days by measuring pain intensity at 6-time points per day using a 10-cm visual analog scale. Cluster analysis was performed using 6 variables with standardized visual analog scale values at 6-time points for individual participants to extract groups with similar pain circadian rhythms. The results of the Neuropathic Pain Symptom Inventory and psychological evaluations in each group were compared using the Kruskal–Wallis test. The results revealed 3 clusters with different circadian rhythms of pain. The total and evoked pain subscale Neuropathic Pain Symptom Inventory scores differed among the 3 clusters. The results suggest that a thorough understanding of circadian pain rhythms in chronic pain patients may facilitate the performance of activities of daily living and physical exercise from the perspective of pain management.     

Microtubules modulate melatonin receptors involved in phase-shifting circadian activity rhythms: in vitro and in vivo evidence

Abstract:  MT1 melatonin receptors expressed in Chinese hamster ovary (CHO) cells remain sensitive to a melatonin re-challenge even following chronic melatonin exposure when microtubules are depolymerized in the cell, an exposure that normally results in MT1 receptor desensitization. We extended our findings to MT2 melatonin receptors using both in vitro and in vivo approaches. Using CHO cells expressing human MT2 melatonin receptors, microtubule depolymerization prevents the loss in the number of high potency states of the receptor when compared to melatonin-treated cells. In addition, microtubule depolymerization increases melatonin-induced PKC activity but not PI hydrolysis via Gi proteins similar to that shown for MT1Rs. Furthermore, microtubule depolymerization in MT2-CHO cells enhances the exchange of GTP on Gi-proteins using a photoaffinity analog of GTP. To test whether microtubules are capable of modulating melatonin-induced phase-shifts, microtubules are depolymerized specifically within the suprachiasmatic nucleus of the hypothalamus (SCN) of the Long Evans rat and the efficacy of melatonin to phase shift their circadian activity rhythms was assessed and compared to animals with intact SCN microtubules. We find that microtubule depolymerization in the SCN using either Colcemid or nocodazole enhances the efficacy of 10 p m melatonin to phase-shift the activity rhythms of the Long Evans rat. No enhancement occurs in the presence of β-lumicolchicine, the inactive analog of Colcemid. Taken together, these data suggest that microtubule dynamics can modulate melatonin-induced phase shifts of circadian activity rhythms which may explain, in part, why circadian disturbances occur in individuals afflicted with diseases associated with microtubule disturbances.