大鼠松果体Clock基因和芳烷脘N-乙酰基转移酶基因的昼夜节律性表达及光照影响

探讨12h光照、12h黑暗交替(12h-light：12h-dark cycle,LD)及持续黑暗(constant darkness,DD)光制下松果体Clock基因和芳烷脘N-乙酰基转移酶基因(arylalkylamine N-acetyltransferase gene,NAT)是否存在昼夜节律性表达及其光反应变化。Sprague-Dawley大鼠在LD和DD光制下分别被饲养4周(n=36)和8周(n=36)后,在一昼夜内每隔4h采集一组松果体组织(n=6),提取总RNA,用竞争性定量RT-PCR测定不同昼夜时点样品中Clock及NAT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。结果如下：(1)在DD或LD光制下,松果体Clock和NAT基因mRNA的表达均呈现夜高昼低的节律性振荡(P＜0．05)。(2)与DD光制下比较,LD光制下松果体Clock和NAT基因的表达振幅及峰值相的mRNA水平均降低(P＜0．05)。(3)在DD或LD光制下,Clock和NAT基因之间显示相似的节律性表达(P＞0．05)。结果表明,Clock和NAT基因在松果体中存在同步的内源性昼夜节律表达,光照作用可使其表达下调。     

Neural Regulation of Dark-Induced Abundance of Arylalkylamine N-Acetyltransferase (AANAT) and Melatonin in the Carp (Catla catla) Pineal: An In Vitro Study

In all the vertebrates, synthesis of melatonin and its rhythm-generating enzyme arylalkylamine N-acetyltransferase (AANAT) reaches its peak in the pineal during the night in a daily light-dark cycle, but the role of different neuronal signals in their regulation were unknown for any fish. Hence, the authors used specific agonist and antagonists of receptors for different neuronal signals and regulators of intracellular calcium (Ca²⁺) and adenosine 3',5'-cyclic monophosphate (cAMP) in vitro to study their effects on the abundance of AANAT and titer of melatonin in the carp (Catla catla) pineal. Western blot analysis followed by quantitative analysis of respective immunoblot data for AANAT protein, radioimmunoassay of melatonin, and spectrophotometric analysis of Ca²⁺ in the pineal revealed stimulatory effects of both adrenergic (α₁ and β₁) and dopaminergic (D₁) agonists and cholinergic (both nicotinic and muscarinic) antagonists, inhibition by both adrenergic and dopaminergic antagonists and cholinergic agonists, but independent of the influence of any agonists or antagonists of α₂-adrenergic receptors. Band intensity of AANAT and concentration of melatonin in the pineal were also enhanced by the intracellular calcium-releasing agent, activators of both calcium channel and adenylate cyclase, and phophodiesterase inhibitor, but suppressed by inhibitor of calcium channel and adenylate cyclase as well as activator of phophodiesterase. Moreover, an inhibitory effect of light on the pineal AANAT and melatonin was blocked by both cAMP and proteasomal proteolysis inhibitor MG132. Collectively, these data suggest that dark-induced abundance of AANAT and melatonin synthesis in the carp pineal are a multineuronal function, in which both adrenergic (α₁ and β₁, but not α₂) and dopaminergic signals are stimulatory, whereas cholinergic signals are inhibitory. This study also provides indications, though arguably not conclusive evidence, that in either case the neuronal mechanisms follow a signal-transduction pathway in which Ca²⁺ and cAMP may act as the intracellular messengers. It also appears that proteasomal proteolysis is a conserved event in the regulation of AANAT activity in vertebrates. (Author correspondence: dgp_skmaitra@yahoo.co.in)     

Gonadal hormones provide the biological basis for sex differences in behavioral responses to cocaine

Both clinical and rodent studies show sexually dimorphic patterns in the behavioral response to cocaine in all phases of the addiction process (induction, maintenance, and relapse). Clinical and rodent studies also indicate that hormonal fluctuations during the menstrual/estrous cycle modulate cocaine-induced subjective effects in women and locomotor activity in female rats. Evidence suggests that gonadal hormones underlie these observed differences and could be the biological basis of sex-specific differences in cocaine addiction. To study the effects of gonadal hormones on cocaine-induced activity, two approaches have been used. First, studies have examined the role of endogenous hormones through gonadectomy (GDX) and side-by-side comparisons with intact rats. Second, the individual contributions of testosterone, progesterone, and estrogen have been determined by hormone replacement in GDX rats. In this review, we discuss gonadal hormones as the biological basis for the behavioral responses to cocaine, and the clinical implications of these findings.     

Orexin A in the VTA is critical for the induction of synaptic plasticity and behavioral sensitization to cocaine

Dopamine neurons in the ventral tegmental area (VTA) represent a critical site of synaptic plasticity induced by addictive drugs. Orexin/hypocretin-containing neurons in the lateral hypothalamus project to the VTA, and behavioral studies have suggested that orexin neurons play an important role in motivation, feeding, and adaptive behaviors. However, the role of orexin signaling in neural plasticity is poorly understood. The present study shows that in vitro application of orexin A induces potentiation of N-methyl-D-aspartate receptor (NMDAR)-mediated neurotransmission via a PLC/PKC-dependent insertion of NMDARs in VTA dopamine neuron synapses. Furthermore, in vivo administration of an orexin 1 receptor antagonist blocks locomotor sensitization to cocaine and occludes cocaine-induced potentiation of excitatory currents in VTA dopamine neurons. These results provide in vitro and in vivo evidence for a critical role of orexin signaling in the VTA in neural plasticity relevant to addiction.     

Circadian locomotor rhythms in the desert iguana I. The role of the eyes and the pineal

Summary The pineal and the eyes are known to be important components in the circadian system of some species of lizards; their effects may be mediated by the hormone melatonin. We examined the role played by these structures in the desert iguana (Dipsosaurus dorsalis). Surgical removal of the pineal had no effect on circadian locomotor rhythms, even though this procedure abolished the circadian rhythm of melatonin in the blood. Furthermore, when the isolated pineal of Dipsosaurus was studied in organ culture, it showed no circadian rhythm of melatonin secretion, as do pineals of some other lizard species, although it did produce large quantities of this hormone. Bilateral ocular enucleation had only small effects on the freerunning period of locomotor rhythms, without affecting melatonin levels in the blood. Behavioral circadian rhythms persisted in desert iguanas subjected to both enucleation and pinealectomy. These data suggest that neither the pineal nor the eyes are central components of the circadian pacemaking system in Dipsosaurus, nor is melatonin critically involved in maintaining its organization.Abbreviations CT circadian time - ZT zeitgeber time - LL constant light - LD light-dark cycle - DD constant darkness - freerunning circadian period     

Zeitgeber Hierarchy in Humans: Resetting the Circadian Phase Positions of Blind People Using Melatonin

Four blind individuals who were thought to be entrained at an abnormal circadian phase position were reset to a more normal phase using exogenous melatonin administration. In one instance, circadian phase was shifted later. A fifth subject who was thought to be entrained was monitored over four years and eventually was shown to have a circadian period different from 24 h. These findings have implications for treating circadian phase abnormalities in the blind, for distinguishing between abnormally entrained and free‐running blind individuals, and for informing the debate over zeitgeber hierarchy in humans.     

On the role of serotonin2A/2C receptors in the sensitization to cocaine.

Apart from showing involvement of dopamine, recent studies also indicate a role of serotonin (5-HT) in the behavioral effects of cocaine in rodents. In the present study we investigated the role of 5-HT2A/2C receptors in the development or expression of sensitization to cocaine in rats, using ketanserin, an antagonist at these receptors. Since ketanserin also shows a high affinity for alpha1-adrenoceptors, prazosin, a comparative antagonist at those receptors was also examined. Male Wistar rats were treated repeatedly (for 5 days) with cocaine (10 mg/kg) in combination with either vehicle, or ketanserin (1-3 mg/kg) or prazosin (3 mg/kg); afterwards, on day 10, they received a challenge dose of cocaine (10 mg/kg). In another experiment, the animals were given either with vehicle or cocaine (10 mg/kg) for 5 days, and were then challenged with cocaine (10 mg/kg) in combination with vehicle, or ketanserin (1-3 mg/kg) or prazosin (3 mg/kg) on day 10. Acute administration of cocaine increased the locomotor activity in rats; that hyperactivation was inhibited by ketanserin (3 mg/kg), but not by prazosin. In animals treated repeatedly with cocaine, the locomotor hyperactivity induced by a challenge dose of the psychostimulant was ca. 2-3 times higher than that after its first administration. No difference was observed in the response to cocaine challenge in rats treated repeatedly with cocaine, ketanserin+cocaine, or prazosin+cocaine. In animals treated repeatedly with the psychostimulant, the behavioral response to a challenge dose of cocaine was dose-dependently decreased when the drug was combined with ketanserin, but not with prazosin. The above findings indicate a role of 5-HT2A/2C receptors (but not alpha1-adrenoceptors) in the acute locomotor hyperactivity, as well as in the expression (but not development) of cocaine sensitization. Since chronic use of cocaine by humans may lead to psychoses or craving for this drug of abuse, our findings also seem to indicate possible importance of 5-HT2A/2C receptor antagonists in the therapy of cocaine addiction.     

Differential reproductive response to short photoperiod in deer mice: role of melatonin

Summary Inhibitory photoperiod differentially effects reproduction in deer mice (Peromyscus maniculatus nebrascensis). Pituitary-testicular function is arrested in about one-third of short-day exposed males (reproductively responsive mice), while an equal number remain fertile (reproductively nonresponsive mice). Both phenotypes are found in natural populations and their disparate reproductive responses have a genetic basis. To assess whether this difference is attributable to a prepineal/pineal or post-pineal mechanism, we compared spermatogenic responses of known and unknown phenotype to exogenous melatonin. Melatonin significantly reduced mean sperm number in long-day housed mice of unknown phenotype. But, individual responses ranged from azoospermia to normal spermatogenesis, and this range was not significantly different from that previously recorded for short-day exposed mice. Reproductively nonresponsive males were unaffected by melatonin administration when housed under long or short daylength. In contrast, melatonin significantly suppressed sperm production in reproductively responsive males housed under long photoperiod, but had no additional suppressive effect in short-day housed mice with regressed testes. These data demonstrate that melatonin is only effective in eliciting testicular regression in reproductively responsive males. Taken together, these results suggest that differential testicular response to photoperiod are caused by a post-pineal mechanism.Abbreviations LD long day - SD short day - 16L:8D 16 h light, 8 h dark - 8L:16D 8 h light, 16 h dark     

氯胺酮诱导不同年龄和性别小鼠的过度活动

虽然氯胺酮(Ketamine)在历史上作为麻醉剂用于人类和家畜,但由于其分解特性, 作为一种娱乐药物似乎更具知名度.以前的研究表明相对于成年人, 孩子使用氯胺酮不易显示出不利影响,但是对其发生的机理几乎没有研究.本文研究了氯胺酮对小鼠的活动程度和固有行为的作用.结果表明:使用氯胺酮可增加22、35和50日龄小鼠的运动器官的敏捷性,并证明了氯胺酮的作用随年龄的增长而降低;使用氯胺酮所导致的旋转与年龄的变化有关,但站立的减少与年龄无关,这种减少不依赖于小鼠的年龄.     

Circadian rhythms of oviposition and feeding activity in Japanese quail: effects of cyclic administration of melatonin

The aim of these experiments was to test the effect of a cyclic administration of melatonin, by mimicking the daily rhythm of hormone levels, on the circadian organization of two distinct functions in quail: oviposition and feeding activity. Laying and feeding rhythms under photoperiodic conditions and constant darkness (DD) were investigated. Under DD, where the two rhythms were free running, a daily rhythm of melatonin was administered. In LD 14h:10h, two different individual profiles of laying were established, with stable females laying at the same time each day and delayed females laying progressively later each day. For feeding activity, all birds were clearly synchronized to the photoperiodic cycle. In DD, the laying birds showed a free-running rhythm of oviposition with a period longer than 24 h for both profiles but the delayed profile females had a longer period than stable profile females. In comparison, the free-running period of feeding rhythm of the same birds was shorter than 24 h. A cyclic administration of melatonin had no effect on laying rhythm, which continued to free-run in DD, whereas feeding activity was synchronized as soon as the first cycle of melatonin was administered. From these results, it seems that two different circadian systems drive each of the two types of behavior separately. Melatonin could be the main synchronizer for the temporal control of feeding behavior, but it does not play a part in the control of oviposition in Japanese quail.     

Sex differences in the response to influenza virus infection: modulation by stress

Influenza virus infection is a significant public health problem; however factors affecting the incidence and severity of disease have not been fully elucidated. The present study sought to examine the role of sex and stress in mediating susceptibility to an influenza viral infection in mice. Male and female mice underwent repeated cycles of restraint (RST) stress, followed by an influenza A/PR8 virus infection. Following these manipulations, levels of circulating corticosterone, lung proinflammatory cytokine gene expression and sickness behavior were examined. The data indicate sex differences in several aspects of the response to the A/PR8 virus infection. The kinetics of lung interleukin-1β mRNA expression were faster in infected males compared to females, while circulating corticosterone levels were elevated in infected females, but not in males. Anorexia and reduced saccharin consumption began earlier and symptoms were more pronounced in infected males than in females. In addition, RST modulated the response to the A/PR8 virus infection. Proinflammatory cytokine gene expression in response to infection was enhanced and sickness behavior was modulated by RST in both males and females. These data suggest that males mount more vigorous immune and behavioral responses to influenza viral infection compared to females, and stress exacerbates the response in both males and females. In conclusion, complex interactions between biological and behavioral factors are involved in mediating individual differences in health and disease. Additional studies may help uncover some of the factors contributing to the individual differences in susceptibility to influenza infection.     

Development of Melatonin Synthesis in Chicken Retina: Regulation of Serotonin N-Acetyltransferase Activity by Light, Circadian Oscillators, and Cyclic AMP

In chicken retinas, melatonin levels and the activity of serotonin N-acetyltransferase (NAT), a key regulatory enzyme of melatonin biosynthesis, are expressed as circadian rhythms with peaks of levels and activity occurring at night. In the present study, NAT activity was examined in retinas of embryonic and posthatch chicks to assess the ontogenic development of regulation of the enzyme by light, circadian oscillators, and the second messenger cyclic AMP. During embryonic development, NAT activity was consistently detectable by embryonic day 6 (E6). Significant light-dark differences were first observed on E20, and increased to a maximum amplitude of sixfold by posthatch day 3 (PH3). Circadian rhythmicity of NAT activity appears to develop at or prior to hatching, as evidenced by day-night differences of activity in constant darkness observed in PH1 chicks that had been exposed to a light-dark cycle in ovo only. NAT activity is regulated by a cyclic AMP-dependent mechanism. Activity was significantly increased by incubating retinas with forskolin or dibutyryl cyclic AMP as early as E7, and seven- to ninefold increases were observed following treatment with these agents on E14. Thus, development of the cyclic AMP-dependent mechanism for increasing NAT activity significantly precedes that of rhythmicity, suggesting that the onset of rhythmicity may be related to the onset of photoreception or development of the circadian oscillator in chick retina.     

Dopamine signaling in food addiction: role of dopamine D2 receptors

Dopamine (DA) regulates emotional and motivational behavior through the mesolimbic dopaminergic pathway. Changes in DA signaling in mesolimbic neurotransmission are widely believed to modify reward-related behaviors and are therefore closely associated with drug addiction. Recent evidence now suggests that as with drug addiction, obesity with compulsive eating behaviors involves reward circuitry of the brain, particularly the circuitry involving dopaminergic neural substrates. Increasing amounts of data from human imaging studies, together with genetic analysis, have demonstrated that obese people and drug addicts tend to show altered expression of DA D2 receptors in specific brain areas, and that similar brain areas are activated by food-related and drug-related cues. This review focuses on the functions of the DA system, with specific focus on the physiological interpretation and the role of DA D2 receptor signaling in food addiction. [BMB Reports 2013; 46(11): 519-526]     

Avian Melatonin Synthesis: Photic and Circadian Regulation of Serotonin N-Acetyltransferase mRNA in the Chicken Pineal Gland and Retina

Abstract: The circadian rhythms in melatonin production in the chicken pineal gland and retina reflect changes in the activity of serotonin N -acetyltransferase (arylalkylamine N -acetyltransferase; AA-NAT; EC 2.3.1.87). Here we determined that the chicken AA-NAT mRNA is detectable in follicular pineal cells and retinal photoreceptors and that it exhibits a circadian rhythm, with peak levels at night. AA-NAT mRNA was not detected in other tissues. The AA-NAT mRNA rhythm in the pineal gland and retina persists in constant darkness (DD) and constant lighting (LL). The amplitude of the pineal mRNA rhythm is not decreased in LL. Light appears to influence the phase of the clock driving the rhythm in pineal AA-NAT mRNA in two ways: The peak is delayed by ∼6 h in LL, and it is advanced by >4 h by a 6-h light pulse late in subjective night in DD. Nocturnal AA-NAT mRNA levels do not change during a 20-min exposure to light, whereas this treatment dramatically decreases AA-NAT activity. These observations suggest that the rhythmic changes in chicken pineal AA-NAT activity reflect, at least in part, clock-generated changes in mRNA levels. In contrast, changes in mRNA content are not involved in the rapid light-induced decrease in AA-NAT activity.     

The Circadian Body Temperature Rhythm in the Elderly: Effect of Single Daily Melatonin Dosing

The present study is part of a more extensive investigation dedicated to the study and treatment of age‐dependent changes/disturbances in the circadian system in humans. It was performed in the Tyumen Elderly Veteran House and included 97 subjects of both genders, ranging from 63 to 91 yrs of age. They lived a self‐chosen sleep‐wake regimen to suit their personal convenience. The experiment lasted 3 wks. After 1 control week, part of the group (n=63) received 1.5 mg melatonin (Melaxen?) daily at 22:30 h for 2 wks. The other 34 subjects were given placebo. Axillary temperature was measured using calibrated mercury thermometers at 03:00, 08:00, 11:00, 14:00, 17:00, and 23:00 h each of the first and third week. Specially trained personnel took the measurements, avoiding disturbing the sleep of the subjects. To evaluate age‐dependent changes, data obtained under similar conditions on 58 young adults (both genders, 17 to 39 yrs of age) were used. Rhythm characteristics were estimated by means of cosinor analyses, and intra‐ and inter‐individual variability by analysis of variance (ANOVA). In both age groups, the body temperature underwent daily changes. The MESOR (36.38±0.19°C vs. 36.17±0.21°C) and circadian amplitude (0.33±0.01°C vs. 0.26±0.01°C) were slightly decreased in the elderly compared to the young adult subjects (p<0.001). The mean circadian acrophase was similar in both age groups (17.19±1.66 vs. 16.93±3.08 h). However, the inter‐individual differences were higher in the older group, with individual values varying between 10:00 and 23:00 h. It was mainly this phase variability that caused a decrease in the inter‐daily rhythm stability and lower group amplitude. With melatonin treatment, the MESOR was lower by 0.1°C and the amplitude increased to 0.34±0.01°C, a similar value to that found in young adults. This was probably due to the increase of the inter‐daily rhythm stability. The mean acrophase did not change (16.93 vs. 16.75 h), although the inter‐individual variability decreased considerably. The corresponding standard deviations (SD) of the group acrophases were 3.08 and 1.51 h (p<0.01). A highly significant correlation between the acrophase before treatment and the phase change under melatonin treatment indicates that this is due to a synchronizing effect of melatonin. Apart from the difference in MESOR, the body temperature rhythm in the elderly subjects undergoing melatonin treatment was not significantly different from that of young adults. The data clearly show that age‐dependent changes mainly concern rhythm stability and synchronization with the 24 h day. A single daily melatonin dose stabilizes/synchronizes the body temperature rhythm, most probably via hypothermic and sleep‐improving effects.     

Trichinella spiralis: role of non-H-2 genes in resistance to primary infection in mice

Two strains of mice which share identical H-2 genes but differ in their genetic backgrounds were compared for their ability to resist infection with Trichinella spiralis. The two strains of mice, C3HeB/FeJ and AKR/J, share the H-2k haplotype which is associated with susceptibility to primary infection with T. spiralis in H-2 congenic strains of mice. AKR/J mice, infected with 150 infective muscle larvae, harbored significantly fewer muscle larvae 30 days postinfection than did mice of the strain C3HeB/FeJ. Approximately equal numbers of worms establish in the small intestine of AKR and C3H mice, but the AKR mice expelled adult worms from the gut more rapidly than did mice of the C3H strain. By Day 9 postinfection, 50% of the worms had been expelled by the AKR mice whereas expulsion of worms from C3H mice was delayed beyond Day 9 and occurred primarily between Days 10 and 12. Over this same experimental period (Days 6-12), fecundity of female worms from AKR mice, measured as the mean newborn larvae/female/hour, was approximately one-half that of worms taken from C3H mice. These results support the conclusion that genes outside of the mouse H-2 complex regulate expulsion of adult worms from the gut. These background genes also markedly influence the fecundity of female worms.     

Site and mechanism of behavioral tolerance to cocaine: A study of dopamine release in Wistar-Kyoto and spontaneously hypertensive rats

Wistar-Kyoto and spontaneously hypertensive rats received i.v. infusions of cocaine hydrochloride (60 mg/kg per day) for 3, 7, and 14 days, or saline for 7 days. Acute cocaine challenge (40 mg/kg, s.c.) was given to treated and control rats 24 hr after the termination of each infusion period. There were no strain differences in brain levels of cocaine during cocaine infusion, nor after cocaine challenges. There were no strain differences in resting levels of [³H]dopamine release. Release of [³H]dopamine decreased in nuclei accumbens of 7- and 14-day cocaine-infused animals. Release of [³H]dopamine was maximal in both brain regions 2 hr after acute cocaine challenge. After 14 days of cocaine infusion, cocaine challenge in both strains reduced [³H]dopamine release in the nucleus accumbens, but not in the striatum; the reduction being greater in Wistar-Kyoto rats. The behavioral tolerance which accompanies similar cocaine infusion regimens may be related to striatal tolerance to cocaine-induced dopamine release.