5-HT_(1A)受体蛋白在PC12细胞膜转运的动态变化

目的在神经元样PC12活细胞上进行实时、可视和定量研究5-羟色胺1A受体的时空分布、膜转运和信号传导机制。方法运用RT-PCR方法获得小鼠5-HT1A基因,并插入到pEGFP-N1真核表达载体中。采用阳离子脂质体方法将质粒转染至PC12细胞和HEK293细胞中,通过G418筛选出稳定表达5-HT1A-EGFP的PC12细胞系。运用激光共聚焦成像系统观察活细胞中5-HT1A-EGFP的表达情况,利用光漂白荧光恢复(FRAP)技术在PC12细胞膜局部漂白后观察5-HT1A-EGFP荧光蛋白在细胞膜上转运的情况。结果克隆所获得的小鼠5-HT1A基因是准确的。5-HT1A-EGFP蛋白清晰的分布于PC12和HKE293细胞膜上。通过FRAP技术观察到漂白区域的细胞膜在100s内有部分恢复,说明受体在细胞膜上发生转运。结论建立了稳定表达5-HT1A-EGFP融合蛋白的PC12细胞系,并利用活细胞成像和FRAP技术观察分析并证实了5-HT1A受体在PC12细胞的膜表面的表达和转运的动态变化。     

Chronic fluoxetine administration desensitizes the hyperglycemia but not the anorexia induced by serotonin in rats receiving fructose-enriched chow

The present study examined the effect of chronic fluoxetine administration on the feeding behavior of fructose-fed rats. Male fructose-fed rats were divided into two groups: (1) control fructose-fed rats (CFR), and (2) fructose-fed rats treated with oral fluoxetine 5 mg/kg/day for 30 days (FFR). The feeding behaviors and plasma glucose levels in response to either serotonin (5-HT, 5 mg/kg) or saline injection were studied. The results showed 5-HT increased CFR plasma glucose in a dose-dependent fashion while FFR demonstrated significantly lower responses to 5-HT stimulation. 5-HT significantly increased the feeding latency and decreased the amount of food intake in the CFR. Fluoxetine treatment did not affect the 5-HT effect on food intake amount but significantly reduced the 5-HT effect on feeding latency. Putting the animals in a new environment increased the 5-HT effect on feeding latency further; the effect was ameliorated in the FFR. In conclusion, 5-HT induced hyperglycemia, increased feeding latency and decreased food intake amount in fructose-fed rats. Chronic administration of fluoxetine counteracted the 5-HT effects on blood glucose level and feeding latency, but not on the amount of food intake.     

Photoreceptor ubiquitination by COP1 E3 ligase desensitizes phytochrome A signaling

Desensitization of activated receptors is an important mechanism for terminating signal transduction. Here we show that phytochrome (phy) A, a predominant photoreceptor for seedling deetiolation, colocalizes in nuclear bodies with CONSTITUTIVELY PHOTOMORPHOGENIC (COP) 1, a RING motif-containing E3 ligase. The phyA PAS domain interacts with the COP1 WD40 domain. Both the Pr and the Pfr forms of phyA, as well as the PHYA apoprotein, are ubiquitinated by COP1 in vitro. The phyA destruction rate is decreased in cop1 mutants and by expression of a COP1 RING motif mutant. Our results indicate that COP1 acts as an E3 ligase to regulate phyA signaling by targeting elimination of the phyA photoreceptor itself.     

Studies on sleep/wake effects of serotonin reuptake inhibitors and receptor subtype involvement

SUMMARY  Studies with the serotonin uptake inhibitors zimeldine and alaproclate show biphasic effects on the sleep/wake axis in rats and cats. Zimeldine induced an initial waking response succeeded by a small SWS-2 increase in rats. The waking increase was not blocked by the 5-HT₂ antagonist ritanserin nor by the putative 5-HT_1A antagonist (-)-alprenolol. In cats, zimeldine induced initial behavioural changes which were succeeded by a large SWS-2 increase. Alaproclate gave similar initial responses as zimeldine in both species, and was succeeded by a moderate sleep increase in cats but not in rats. The complex sleep/wake effects following the serotonin uptake inhibitors may result from simultaneous induction of incompatible serotonergic effects.     

A selective serotonin reuptake inhibitor reduces REM sleep in the homing pigeon

Avian and mammalian 'rapid eye movement' sleep (REM sleep) resemble each other in several aspects. However, the question of whether REM sleep has a shared evolutionary ancestry in birds and mammals has yet to be thoroughly explored. The brain regions and neurotransmitter systems involved in the generation of mammalian REM sleep are phylogenetically ancient, and are also found in extant birds and reptiles. Several pharmacological experiments in birds indicate that similar neural substrates are involved in the regulation of avian and mammalian sleep. However, because the drugs used in these studies generally resulted in non-specific sleep loss, the neurochemical regulation of avian REM sleep in particular remains uncertain. The selective serotonin reuptake inhibitor (SSRI) zimelidine is known to reduce REM sleep in mammals. If avian REM sleep is similarly regulated by serotonin, it would be expected that an acute dose of a SSRI should also reduce avian REM sleep. To investigate a putative role of serotonin in the regulation of avian REM sleep, changes in sleep electroencephalogram (EEG) and behavior were recorded in five pigeons (Columba livia) after the administration of an acute dose of zimelidine. Our results demonstrate that the effects of zimelidine on avian REM sleep are comparable to those observed in mammals, indicating that serotonin may serve a similar function in the control of avian and mammalian REM sleep.     

Effect of serotonin 5-HT1A receptor agonists on sexual motivation of male mice

The effect of selective agonists of serotonin 5-HT_1A receptors on sexual arousal (its behavioral and hormonal components) in male CBA mice caused by estral females was studied. Injections of 8-OH-DPAT, flesinoxane, and ipsapirone significantly decreased the main behavioral parameter of sexual motivation (duration of the male's stay near the wall separating it from the receptive female). The activating effect of the female on the pituitary gonadal system of the male was completely blocked: the blood testosterone level did not increase. Therefore, the behavioral and hormonal components of sexual activation of males are regulated by the serotonin mechanisms alone, in which the cerebral 5-HT_1A receptors are involved. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 2, pp. 224–226, February, 1999     

SHP2 forecast for the immune system: fog gradually clearing

The src homology 2 (SH2) domain containing tyrosine phosphatase SHP2 (also referred to as SHP-2) is ubiquitously expressed in mammalian tissues and has been shown to be essential for embryonic development, haematopoiesis and signalling downstream of a variety of growth factors. Dysregulation of SHP2 function or expression has recently been implicated in the pathogenesis of human diseases involving haematopoietic cell lineages. New findings also demonstrate the involvement of SHP2 in the regulation of immune responses through its effects on cytokine and inhibitory receptor signalling pathways, and novel transgenic models are providing valuable insights into the role of SHP2 in T cells.     

Total sleep deprivation increases extracellular serotonin in the rat hippocampus

Sleep deprivation exerts antidepressant effects after only one night of deprivation, demonstrating that a rapid antidepressant response is possible. In this report we tested the hypothesis that total sleep deprivation induces an increase in extracellular serotonin (5-HT) levels in the hippocampus, a structure that has been proposed repeatedly to play a role in the pathophysiology of depression. Sleep deprivation was performed using the disk-over-water method. Extracellular levels of 5-HT were determined in 3 h periods with microdialysis and measured by high performance liquid chromatography coupled with electrochemical detection. Sleep deprivation induced an increase in 5-HT levels during the sleep deprivation day. During an additional sleep recovery day, 5-HT remained elevated even though rats displayed normal amounts of sleep. Stimulus control rats, which had been allowed to sleep, did not experience a significant increased in 5-HT levels, though they were exposed to a stressful situation similar to slee-deprived rats. These results are consistent with a role of 5-HT in the antidepressant effects of sleep deprivation.     

Seasonality associated with the serotonin 2A receptor -1438 A/G polymorphism

BACKGROUND: Seasonal affective disorder and seasonal rhythms in mood and behavior (seasonality) have been reported to be associated with serotonergic system. In this study we investigated the relationship between the serotonin 2A receptor (5HTR2A) -1438 A/G polymorphism and seasonal variation in a young Korean healthy population. METHODS: 297 young Korean medical students were recruited for this study. They were genotyped for the 5HTR2A -1438 A/G polymorphism and evaluated the seasonal variation in mood and behavior by the Seasonality Pattern Assessment Questionnaire (SPAQ). RESULTS: The Global Seasonality Score of the SPAQ among three genotypes were not different. However, the comparison between seasonals and non-seasonals showed significant difference in the genotype distribution. The winter-type seasonals showed a significantly higher frequency of the 5HTR2A -1438 A allele compared with non-seasonals (chi2 = 6.80, p = 0.009; OR = 1.79; 95% CI, 1.15-2.78). CONCLUSION: These results suggest that the 5HTR2A -1438 A/G polymorphism is possibly related to seasonality in the Korean population.     

Interleukin 1 receptor contributes to methamphetamine- and sleep deprivation-induced hypersomnolence

Methamphetamine-induced wakefulness is dependent on monoamine transporter blockade. Subsequent to methamphetamine-induced wakefulness, the amount of time spent asleep and the depth of sleep are increased relative to baseline sleep. The mechanisms that drive methamphetamine-induced hypersomnolence are not fully understood. We recently observed that methamphetamine exposure elevates the expression of the sleep-promoting cytokine, interleukin-1β in CD11b-positive monocytes within the brain. Here, we sought to determine whether activation of the interleukin 1 receptor (IL1R) drives the increase in the depth and amount of sleep that occurs subsequent to methamphetamine-induced wakefulness. IL1R-deficient mice and wild type control mice were subjected to systemic methamphetamine (1 and 2mg/kg) and saline treatments. The wake-promoting effect of methamphetamine was modestly potentiated by IL1R-deficiency. Additionally, the increase in time spent in NREMS subsequent to methamphetamine-induced wakefulness in wild type mice was abolished in IL1R-deficient mice. The increase in time spent asleep after 3h of behaviorally enforced wakefulness was also abolished in IL1R-deficient mice. Increases in EEG slow wave activity triggered by methamphetamine and sleep deprivation were of equal magnitude in IL1R-deficient and wild type mice. These data demonstrate that IL1R activation contributes to hypersomnolence that occurs after sleep loss, whether that sleep loss is triggered pharmacologically by methamphetamine or through behavioral sleep deprivation.     

Increased DNA methylation status of the serotonin receptor 5HTR1A gene promoter in schizophrenia and bipolar disorder

Background

Epigenetic changes may play a role in the etiology of psychotic diseases. It has been demonstrated that the serotonin receptor, 5HTR1A, is implicated in schizophrenia (SCZ) and bipolar disorder (BPD). The aim of this study was to investigate the methylation status of a promoter region of the 5HTR1A gene in BPD and SCZ patients.

Methods

Our study included 58 BPD and 40 SCZ (DSM-IV criteria) as well as 67 control subjects. DNA was extracted from blood leukocytes and high-resolution melt (HRM) method was used for analysis.

Results

Non-parametric analysis of variance (Kruskal-Wallis) within groups was significant: H = 67.6; p < 0.0001. The Mann-Whitney U-test showed increased methylation level in both BPD (Z = − 7.4; p < 0.0001) and SCZ (Z = 4.2; p < 0.0001) compared to controls. No effect either of age or gender by own, was observed. ANCOVA revealed a modest effect of age/gender covariance (F = 3.99; p < 0.048).

Limitation

We used a peripheral tissue. The relationship between methylation of blood and brain DNA is not well known. Data need to be replicated in a brain tissue.

Conclusion

We observed increased DNA methylation in the promoter region of the 5HTR1A gene of SCZ and BPD. This could explain the reported decrease of the receptor expression. The current study supports the growing interest of DNA methylation in psychopathology.     

Regulation of dendrite formation of Purkinje cells by serotonin through serotonin1A and serotonin2A receptors in culture

Serotonergic fibers and receptors appear in the rat cerebellum during early postnatal development. In the present study, we investigated the actions of serotonin (5-HT) and its receptors in the dendrite formation of Purkinje cells in organotypic cultures of anterior and posterior lobes of the cerebellum at postnatal day 7. In anterior lobes after 4 days in vitro (4DIV), the dendritic areas and branchings of Purkinje cells were increased by the treatment of 2 microM 5-HT, but decreased by 20 microM 5-HT. In posterior lobes after 4DIV, the dendritic areas of Purkinje cells were increased by 5-HT (2, 20 and 200 microM). In contrast, 5-HT treatment decreased dendritic areas of Purkinje cells in both anterior and posterior lobes after 7DIV. Next, we determined the actions of specific 5-HT receptors in mediating the effects of 5-HT by treatment with selective 5-HT receptor agonists. In anterior lobes after 4DIV, dendritic areas of Purkinje cells were increased by a 5-HT1A receptor agonist (8-OH-DPAT), whereas decreased by a 5-HT2A receptor agonist (DOI). The present study suggested that the dendrite formation of Purkinje cells is promoted by 5-HT through 5-HT1A receptors, but inhibited by 5-HT through 5-HT2A receptors.     

Differential distribution of MAP1A isoforms in the adult mouse barrel cortex and comparison with the serotonin 5-HT2A receptor

Microtubule-associated protein 1A (MAP1A) is essential during the late differentiation phase of neuronal development. Here, we demonstrated the presence of two MAP1A isoforms with a differential spatial distribution in the adult mouse barrel cortex. Antibody A stained MAP1A in pyramidal and stellate cells, including dendrites that crossed layer IV in the septa between barrels. The other antibody, BW6 recognized a MAP1A isoform that was mainly confined to the barrel hollow and identified smaller caliber dendrites. Previously, an interaction of MAP1A and the serotonin 5-hydroxytryptamine 2A (5-HT(2A)) receptor was shown in the rat cortex. Here, we identified, by double-immunofluorescent labeling, MAP1A isoform and serotonin 5-HT(2A) receptor distribution. MAP1A co-localized mainly with 5-HT(2A) receptor in larger apical dendrites situated in septa. This differential staining of MAP1A and a serotonin receptor in defined barrel compartments may be due to changes in the expression or processing of MAP1A during dendritic transport as a consequence of functional differences in processing of whisker-related sensory input.     

Dose-dependent effects of the 5-HT1A receptor agonist 8-OH-DPAT on sleep and wakefulness in the rat

SUMMARY  Sleep and wakefulness were studied in rats following administration of a selective 5-HT_1A agonist (8-OH-DPAT), a non-selective 5-HT_1A antagonist [(-) pindolol] and a combination of 8-OH-DPAT and (—) pindolol.
8-OH-DPAT (1.0–4.0 μg) injected into the dorsal raphe nucleus increased slow-wave sleep and decreased wakefulness. Administration of the 5-HT_1A agonist by subcutaneous route induced biphasic effects such that low doses (0.010 mg kg^-1) decreased wakefulness and increased slow-wave sleep while higher doses (0.375 mg kg^-1) induced opposite effects. REM sleep was suppressed and REM latency was increased, what could be tentatively ascribed to a non-specific effect (hypothermia). (-) Pindolol (1.0–4.0 mg kg^-1) induced an initial increase of wakefulness and a decrease of NREM sleep and REM sleep. Thereafter, NREM sleep showed a marked increase while REM sleep remained depressed. Pretreat-ment with (—) pindolol reversed the effects of both small and large doses of 8-OH-DPAT on slow-wave sleep and wakefulness.
The opposite effects, observed on the waking EEG after activation of either serotonin autoreceptors or postsynaptic 5-HT_1A receptors with adequate doses of 8-OH-DPAT, tend to indicate an active role for the 5-HT_1A receptor in the control of the waking state.     

5-HT3A serotonin receptor in the gastrointestinal tract: the link between immune system and enteric nervous system in the digestive form of Chagas disease

Chagas disease is caused by Trypanosoma cruzi and remains one of the most neglected diseases in Latin America. One of its clinical forms is Chagas megacolon. Despite being known for more than half a century, detailed causes are still obscure. Recent evidence indicates a close relationship between the immune system and the enteric nervous system in the etiology of chagasic megacolon pathology. It is believed that low expression of the 5-HT_3A serotonin receptor on lymphocytes could be linked to megacolon development. To test this hypothesis, this work investigated the distribution of CD4, CD8, and CD20 lymphocytes and their 5-HT_3A receptor expression. The results demonstrated that Chagas patients without megacolon present a higher expression of the 5-HT_3A receptor in all analyzed lymphocytes compared with Chagas patients with megacolon. These data suggest that the high expression of this receptor may lead to immunomodulation and prevent the development of Chagas megacolon.

     

A serotonin 5-HT1A receptor agonist prevents behavioral sensitization to L-DOPA in a rodent model of Parkinson's disease

Marked fluctuation of dopamine concentration in the striatum following long-term L-DOPA administration contributes to the development of L-DOPA-induced motor complications including L-DOPA-induced dyskinesias and wearing-off in patients with Parkinson's disease. We have shown that pretreatment with 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT1A (5-hydroxytryptamine) receptor agonist, alleviates fluctuation of dopamine levels in the dopamine-denervated striatum of 6-hydroxydopamine-lesioned (hemiparkinsonian) rats after L-DOPA treatment. To determine whether co-administration of 8-OH-DPAT with L-DOPA prevents L-DOPA-induced motor complications, we examined rotation behavior and levels of messenger RNAs coding for dynorphin and glutamic acid decarboxylase in the striatum of 6-hydroxydopamine-lesioned rats treated with L-DOPA alone or L-DOPA + 8-OH-DPAT, twice daily, for 2 weeks. Co-administration of 8-OH-DPAT inhibited an increase of rotation behavior to L-DOPA and L-DOPA-induced increases in levels of messenger RNAs coding for dynorphin and glutamic acid decarboxylase in the dopamine-denervated striatum, both of which are established indices of L-DOPA-induced motor complications. These results suggest that pharmaceutical products that stimulate 5-HT1A receptors could prove useful in prevention of the development of L-DOPA-induced motor complications in patients with Parkinson's disease.