Perforant path kindling induces differential alterations in the mRNA levels coding for prodynorphin and proenkephalin in the rat hippocampus

The effect of perforant path kindling on the levels of mRNAs coding for proenkephalin and prodynorphin in hippocampus and frontal cortex of rats was measured using RNA blot analysis. In rats showing stage 3 kindled seizures, after consecutive stimulation of the right perforant path, a decrease in the level of prodynorphin mRNA and an increase in levels of proenkephalin mRNA in the ipsilateral hippocampus was found. In addition, the levels of prodynorphin were also decreased in the contralateral hippocampus. No changes in the opioid peptide mRNAs were found in the frontal cortex of the animals. The altered mRNA levels in the hippocampus returned to normal 8 days following cessation of the electrical stimulation. However, at that time a single stimulus was still effective in producing stage 3 kindling seizures. These findings indicate that (1) the opioid peptide gene expression in the hippocampus can be transynaptically altered by kindling of the perforant path and (2) that the opioid peptides may play a role in the development, but not in the maintenance of kindling.     

大鼠海马tau蛋白过度磷酸化时细胞周期蛋白cyclin D1的表达

目的:探讨大鼠海马tau蛋白过度磷酸化时细胞周期蛋白cyclin D1的表达.方法:用cAMP-依赖性蛋白激酶 A (PKA) 的激动剂Forskolin注射大鼠侧脑室,免疫印迹和免疫组织化学技术检测大鼠海马tau蛋白的磷酸化水平,同时采用免疫印迹、免疫组织化学和RT-PCR方法检测海马cyclin D1和cyclin D1 mRNA.结果:侧脑室注射Forskolin 48h,大鼠海马tau蛋白Ser214、Ser396和Ser202/Thr205位点的磷酸化水平升高,同时检测到细胞周期蛋白cyclin D1和cyclin D1 mRNA.结论:侧脑室注射Forskolin诱导大鼠海马 tau蛋白过度磷酸化的同时,大鼠海马出现细胞周期蛋白cyclin D1的表达,提示了tau蛋白过度磷酸化与细胞周期相关蛋白cyclin D1之间可能的内在联系.     

Chlamydia trachomatis infection induces cleavage of the mitotic cyclin B1

The obligate intracellular pathogen Chlamydia trachomatis interferes with a number of host cell processes, including cytoskeletal organization, vesicular trafficking, and apoptosis. In this study we report that C. trachomatis-infected cells proliferate more slowly than uninfected cells, suggesting that C. trachomatis may also manipulate the eukaryotic cell cycle. We further demonstrate that C. trachomatis infection destabilizes specific cell cycle proteins involved in the G2/M transition. C. trachomatis-infected cells, compared to uninfected cells, have lower levels of cyclin-dependent kinase 1. Additionally, C. trachomatis infection induces an N-terminal truncation of the mitotic cyclin B1. Manipulation of the host cell cycle may represent a strategy used by C. trachomatis to ensure a stable environment conducive to bacterial growth and replication.     

Prognostic value of cyclin B1 protein expression in colorectal cancer

We used immunohistochemical analysis to study the expression and prognostic impact of cyclin B1, a key molecule for G2-M phase transition during the cell cycle, in a series of 342 curatively resected colorectal carcinomas. In 269 tumors (78.7%), high expression of cyclin B1 in more than 10% of tumor cells was observed, but there was no association between cyclin B1 expression and histopathologic tumor features. Univariate analysis revealed no impact on disease-free and overall survival. Multivariate analysis revealed pT and pN categories, extramural blood vessel invasion, and low-grade tumor cell differentiation as independent prognostic predictors for overall survival, and pT and pN categories and tumor site for disease-free survival. According to our results, high expression of cyclin B1 is a frequent and early event in colorectal carcinomas. However, cyclin B1 expression is neither a predictor of prognosis in patients with colorectal cancer nor a suitable tool for identifying subgroups of patients at higher risk for disease recurrence.     

Pentylenetetrazol kindling and factors of glutamate transmitter metabolism in rat hippocampus.

Male Wistar rats administered repetitively with pentylenetetrazol developed a dose-dependent enhancement of seizure behaviour referred to as pentylenetetrazol kindling. After a daily dose of 40 mg pentylenetetrazol/kg or physiological saline (control rats) injected intraperitoneally for a period of two weeks, hippocampal tissue was studied autoradiographically for high-affinity uptake of [3H]glutamate and, by activity staining, for aspartate aminotransferase and glutamate dehydrogenase. Most prominent changes were found in neuropil areas known to be endowed with glutamatergic structures. The uptake capacity of glutamate decreased by 48% (maximum rate), whilst activities of aspartate aminotransferase and glutamate dehydrogenase elevated to 140 and 130%, respectively. Cytochrome c oxidase activity was found to be unaffected. The findings indicate an important role of factors of the glutamate metabolism in the kindling process with respect to the production, utilization, and availability of transmitter glutamate.     

Prenatal protracted irradiation at very low dose rate induces severe neuronal loss in rat hippocampus and cerebellum

Prenatal irradiation is known to damage the developing brain. However, little is known about the consequences of very low dose rate prenatal protracted irradiation over several days on neuron numbers in the offspring brain, and on volumes of the corresponding brain regions. Pregnant Wistar rats were exposed either to a protracted γ irradiation from embryonic day (E) 13 to E16 (0.7 mGy/min; total cumulative dose approximately 3 Gy) or were sham-irradiated. Thirty months old male and female offspring were then analyzed for alterations in hippocampal and cerebellar morphology. Using design-based stereology and the analysis of sets of sections systematically and randomly sampled to span the entire brain region of interest, a statistically significant decrease in numbers of hippocampal pyramidal and granule cells as well as of cerebellar Purkinje and granule cells (approximately 50%) was found in male and female irradiated offspring. The volumes of these brain regions were comparably altered. The analysis of only a “representative” section per animal yielded mostly non-significant trends. Evaluation of neuron densities showed no differences between prenatally irradiated and sham-irradiated offspring. Most importantly, very low dose rate prenatal protracted γ irradiation did not result in the same morphologic alterations in the offspring brain as previously observed after prenatal single irradiation such as derangement of the laminar structure of pyramidal cells within the hippocampus or malformation of cerebellar lobules.     

Expression of the neuronal calcium sensor visinin-like protein-1 in the rat hippocampus

Visinin-like protein-1 (VILIP-1) belongs to the neuronal calcium sensor (NCS) family of EF-hand Ca(2+)-binding proteins which are involved in a variety of Ca(2+)-dependent signal transduction processes in neurons. VILIP-1 has been implicated in the pathology of CNS disorders including Alzheimer's disease and schizophrenia, but its expression has also been found to be regulated following induction of hippocampal synaptic plasticity underlying learning and memory processes. VILIP-1 is strongly expressed in different populations of principal and non-principal neurons in the rat hippocampus. VILIP-1-containing interneurons are morphologically and neurochemically heterogeneous. On the basis of co-localizing markers, VILIP-1 is rarely present in perisomatic inhibitory parvalbumin containing cells. However, VILIP-1 is frequently expressed in mid-proximal dendritic inhibitory cells characterized by calbindin immunoreactivity, and most strongly co-expressed in calretinin-positive disinhibitory interneurons. Partial co-localization of the metabotropic glutamate receptor mGluR1alpha with VILIP-1 was often found in interneurons located in the stratum oriens of the hippocampal CA1 region and in hilar interneurons. Partial co-localization of alpha4beta2 nicotinic acetylcholine receptor with VILIP-1 was seen in stratum oriens interneurons and particularly at the border of the hilus in the dentate gyrus, where VILIP-1 also strongly co-localized with calretinin. We speculate that depending on the regulation of the expression of VILIP-1 in hippocampal pyramidal cells or defined types of interneurons, it may have different effects on hippocampal synaptic plasticity and network activity in health and disease.     

Stress induces neuronal death in the hippocampus of castrated rats.

Whereas loss of CA3 neurons in the hippocampus of monkeys which died of stress ulcers suggests that some structural changes may occur, there is no direct evidence that shows stress-induced irreversible changes of neurons. When rats were orchidectomized (castrated) and stressed by restraint and immersion in water for 15 min/day for 30 days, significant loss of hippocampal CA3 and CA4 neurons was observed. Furthermore, primary cultured hippocampal neurons survived shorter when treated with corticosterone. This neuronal loss was prevented by simultaneous administration of testosterone in vivo and in vitro. These findings indicate that stress can contribute to neuronal degeneration associated with hypogonadal conditions such as aging.     

Transient expression of phospholipase D1 in developing rat hippocampus

We investigated the distribution of phospholipase D1 (PLD1) protein in the developing rat hippocampus using an affinity-purified peptide antibody against PLD1. Immunoreactivity for PLD1 was first seen in some scattered cells in the hippocampus at embryonic day 18. At postnatal day 1 (P1), many PLD1 immunoreactive cells were observed in the CA1 and CA3 sectors, subiculum and the hilus of the dentate gyrus. During the first postnatal week, there was an abrupt increase of immunoreactive neurons in the hippocampus, and their number and intensity peaked at P7. During the second postnatal week, there was an abrupt decrease in the number of immunoreactive hippocampal neurons. By P14, no significant labeling was found in the hippocampus. These results corresponded well with those from Western blot analysis, suggesting that PLD1 may regulate the developmental processes of hippocampal neurons.     

Anti-epileptiform effects of audiogenic seizure priming on in vitro kindling in rat hippocampus

The effect of priming for audiogenic seizures (AGS) on the development of epileptiform activity in the hippocampus was studied using in vitro kindling (IVK) in Long-Evans rats. AGS priming consists of intense auditory stimulation during a critical period of auditory development, resulting in sound-induced clonic convulsions upon subsequent testing. Between postnatal day (PND) 28 and 50, slices from subjects primed and sham-primed for AGS on PND 18 were used for recording responses in area CA1 of hippocampus following Schaffer collateral stimulation from stratum radiatum of area CA2/CA3. The developmental priming procedure, which enhances auditory brainstem excitability, resulted in fewer afterdischarges in slices from primed subjects across initial IVK stimulation sequences. These results suggest that changes in excitability that occur with acoustic priming can initially diminish selective epileptiform response characteristics in forebrain areas such as the hippocampus.     

Early maternal deprivation induces alterations in brain-derived neurotrophic factor expression in the developing rat hippocampus

The effects of maternal deprivation (MD) during early postnatal life on the brain-derived neurotrophic factor (BDNF) level were investigated in the present study. Wistar rats were assigned to either maternal deprivation or mother-reared control (MRC) groups. MD manipulation was achieved by separating rat pups from their mothers for 3h a day during postnatal days (PND) 10-15. At 16, 20, 30, and 60 days of age, the level of BDNF mRNA in the hippocampal formation of each group was determined using real-time PCR analysis. Early postnatal maternal deprivation of rat pups resulted in a significant increase in body weight at 60 days of age. The expression of BDNF mRNA in the hippocampus was significantly decreased at 16 days of age, and increased at 30 and 60 days of age. These data indicate that even a brief period of maternal deprivation during early postnatal life can affect hippocampal BDNF expression.     

Corticosterone and phenytoin reduce neuronal nitric oxide synthase messenger RNA expression in rat hippocampus.

The production and release of the corticosteroids, namely the glucocorticoids and the mineralocorticoids, are regulated by various stimuli, including stress. Previous studies from our laboratory have shown that chronic exposure to stress or to stress levels of glucocorticoids produces atrophy of the apical dendrites of CA3 pyramidal neurons in the hippocampus. This stress-induced dendritic remodeling is blocked by the anti-epileptic drug phenytoin, which suppresses glutamate release, and also by N-methyl-D-aspartate receptor antagonists. These results suggest an interaction between glucocorticoids and excitatory amino acids in the development of stress-induced atrophy of CA3 pyramidal neurons. Since nitric oxide is proposed to play an important role in mediating both the physiological and pathophysiological actions of excitatory amino acids, we examined the regulation of neuronal nitric oxide synthase messenger RNA expression by corticosterone and phenytoin in the rat hippocampus. The expression of neuronal nitric oxide synthase messenger RNA in hippocampal pyramidal neurons and granule neurons of the dentate gyrus was unaffected by 21-day administration of corticosterone (40 mg/kg), phenytoin (40 mg/kg) or the combination of corticosterone and phenytoin. However, in hippocampal interneurons, corticosterone/ phenytoin co-administration led to a significant reduction in neuronal nitric oxide synthase messenger RNA levels when compared with vehicle controls. These results suggest that, during exposure to stress levels of corticosterone, phenytoin inhibits glucocorticoid-induced atrophy of CA3 pyramidal neurons by reducing neuronal nitric oxide synthase expression in hippocampal interneurons. Moreover, these results may provide another example of synaptic plasticity in the hippocampus mediated by nitric oxide synthase.     

大鼠血管性痴呆模型动物中海马神经元凋亡和蛋白表达的研究

目的:观察不同时点分别结扎左、右颈总动脉建立大鼠血管性痴呆模型中海马CA1区神经元凋亡和Bcl-2及Bax蛋白表达的影响,探讨其在血管性痴呆发病过程中的作用。方法:采取间隔3 d分2次结扎双侧颈总动脉建立血管性痴呆模型,术后4周用TUNEL法检测海马CA1区神经元凋亡,用免疫组织化学法检测其Bcl-2及Bax蛋白表达。结果:模型组大鼠海马CA1区可见大量凋亡神经元;模型组Bcl-2及Bax蛋白表达明显增加,与假手术组比较差异均有显著意义(P<0.05)。结论:此血管性痴呆模型大鼠中海马CA1区神经元大量凋亡丢失,可能是导致血管性痴呆的病理基础。     

MeCP2 expression and promoter methylation of cyclin D1 gene are associated with cyclin D1 expression in developing rat epididymal duct

Hypermethylation-dependent silencing of the gene is achieved by recruiting methyl-CpG binding proteins (MeCPs). Among the MeCPs, MeCP2 is the most abundantly and ubiquitously expressed in various types of cells. We first screened the distribution and expression pattern of MeCP2 in adult and developing rat tissues and found strong MeCP2 expression, albeit rather ubiquitously among normal tissues, in ganglion cells and intestinal epithelium in the small intestine, in Purkinje cells and neurons in the brain, in spermatogonia and in epithelial cells in the epididymal duct of the testis. We then assessed the expression and the methylation pattern of the promoter region of cyclin D1 by immunohistochemistry and sodium bisulfite mapping, and found that cyclin D1 expression in the epididymal duct decreased rapidly during rat development: strong in newborn rats and very weak or almost negative in 7-day-old rats. Mirroring the decrease of cyclin D1 expression, methylated cytosine at both CpG and non-CpG loci in the cyclin D1 promoter was frequently observed in the epididymal duct of 7-day-old rats but not in that of newborn rats. Interestingly, MeCP2 expression also increased concomitant with the increase of methylation. Cyclin D1 expression in the epididymal duct may be efficiently regulated by the epigenetic mechanism of the cooperative increase of MeCP2 expression and promoter methylation.     

Chronic nicotine and smoking exposure decreases GABA(B1) receptor expression in the rat hippocampus

Nicotine and smoking have long been proved to play an important role in cognition and memory in the hippocampus. This effect is closely related to the gamma-aminobutyric acid (GABA)ergic system. Previous research has focused on functional and pharmacological aspects of nicotine's modulation activity. In this study, the effects of nicotine and different doses of smoking on GABA(B1) expression in the rat hippocampus have been examined using in situ hybridization and RNase protection assay. GABA(B1) receptor mRNAs were intensely expressed in the CA1, CA2, CA3, and dentate gyrus areas of the hippocampus. Nicotine and smoking doses dependently decreased GABA(B1) receptor expression in the hippocampus. These results revealed new aspects of nicotine's modulation on GABA(B) receptor, and on learning and memory.     

Status epilepticus induces time-dependent neuronal and astrocytic expression of interleukin-1 receptor type I in the rat limbic system

Interleukin-1beta is rapidly synthesized by glia after the induction of seizures. Recent evidence shows that endogenous IL-1beta has proconvulsant actions mediated by interleukin-1 receptor type I. This receptor also mediates interleukin-1beta effects on neuronal susceptibility to neurotoxic insults. In this study, we investigated the basal and seizure-induced expression of interleukin-1 receptor type I in rat forebrain to identify the cells targeted by interleukin-1beta during epileptic activity. Self-sustained limbic status epilepticus was induced in rats by electrical stimulation of the ventral hippocampus. Interleukin-1 receptor type I immunoreactivity was barely detectable in neurons in control brain tissue. During status epilepticus, interleukin-1 receptor type I was induced in the hippocampal neurons firstly, and several hours later in astrocytes localized in limbic and extralimbic areas. Neuronal interleukin-1 receptor type I expression in the hippocampus outlasted the duration of spontaneous electroencephalographic seizure and was not observed in degenerating neurons. Astrocytic expression occurred transiently, between six and 18 h after the induction of status epilepticus and was invariably found in regions of neuronal damage. These time-dependent, cell- and region-specific changes in interleukin-1 receptor type I expression during status epilepticus suggest that interleukin-1 receptor type I in neurons mediates interleukin-1beta-induced fast changes in hippocampal excitability while interleukin-1 receptor type I receptors in astrocytes may mediate interleukin-1beta effects on neuronal survival in hostile conditions.     

去神经支配大鼠海马内MTSS1的表达变化

目的:探讨切割穹窿海马伞去神经支配大鼠海马内转移抑制蛋白1(metastasis suppressor 1,MTSS1)的表达变化。方法:SD大鼠随机分成正常组和切割穹窿海马伞后1、3、5、7、14 d组,采用Real-time PCR和免疫荧光组织化学技术观察穹窿海马伞切割后海马内MTSS1 mRNA的表达变化和MTSS1阳性细胞的分布情况。结果:(1)Real-time PCR结果显示:海马内MTSS1 mRNA的相对表达量在切割后3 d开始升高(P<0.05),5 d达到最高水平(P<0.01),7 d恢复至正常水平;(2)免疫荧光组织化学染色结果显示:MTSS1阳性细胞主要表达于海马齿状回门区及颗粒下层中,切割后5 d MTSS1阳性细胞数开始增多(P<0.01),7 d继续增多并达到高峰(P<0.01),14 d时开始下降至正常水平。结论:结合本课题组以往的工作,上述结果提示切割穹窿海马伞后MTSS1的高表达可能与海马神经再生过程中神经干细胞向神经元的分化有关。     

Overexpression of cyclin A and cyclin B1 proteins in astrocytomas

BACKGROUND: Cyclins are proteins that are expressed during the progression of a normal cell through the cell cycle. In a number of cancers, overexpression of cyclin A and cyclin B1 proteins has been reported, and in some instances the levels of expression correlated well with the grades of malignancy. The expression of cyclin A and cyclin B1 proteins in astrocytoma may be linked to the histologic grade or proliferative activities. OBJECTIVE: To study the expression of cyclin A and cyclin B1 proteins in astrocytomas and correlate the labeling indices (LIs) of cyclin A and cyclin B1 with histologic grade and Ki-67 LI. DESIGN: The surgical biopsy specimens from 65 adults with astrocytomas were reviewed and divided into grades based on the World Health Organization system. The paraffin sections were immunostained using primary antibodies against Ki-67, cyclin A, and cyclin B1. The LIs of these astrocytomas for the 3 different antibodies were determined by computerized image analysis. RESULTS: The cyclin A LI showed good correlation with astrocytoma grade and Ki-67 LI. Both the nuclear and cytoplasmic cyclin B LIs correlated well with the tumor grade but showed poor correlation with Ki-67 LI. CONCLUSIONS: This study suggests that although both cyclin A and B protein expression are related to the grade of malignancy in astrocytomas, cyclin A levels more generally reflect the proliferative state of these tumors. We also provide indirect evidence that cyclin B1 is associated with the aberrant progression through the G2-M phase checkpoint in astrocytomas.