Synapses in the granule cell layer of the rat dentate gyrus: serial-sectioning study

Synaptic contacts on the granule cell somata as well as on their axon initial segments in the dentate gyrus of one juvenile 5-week-old rat and one adult 12-week-old rat were analyzed in an electron microscopic serial-sectioning study. In the dentate gyrus of the juvenile rat, somata of 17 granule cells were nearly completely reconstructed from a series of 183 serial sections, and the axon initial segments of 15 of these granule cells were traced in various lengths. On the other hand, in the dentate gyrus of the adult rat, somata of 31 granule cells were almost completely reconstructed from a series of 238 serial sections, and the axon initial segments of 23 of these granule cells were traced in various lengths. Both symmetrical and asymmetrical synapses were observed on the somata, whereas almost all synapses on the axon initial segments were of symmetrical type and asymmetrical synapses on the axon initial segments were rather exceptional. Although we confirmed two conclusions from previous random-section studies to some extent, that is, the superficial-to-deep gradient of synaptic densities on granule cell somata and the presence of a substantial number of asymmetrical synapses on granule cell somata (about 23% of total somatic synapses), the present serial-sectioning study clearly revealed that granule cells vary greatly with regard to the number of synapses on their somata (15–186 in a 5-week-old rat and 9–144 in a 12-week-old rat) and axon initial segments. The granule cells also differed in the proportion of somatic asymmetrical synapses to total synapses they received (0–44% in a 5-week-old rat and 0–60% in a 12-week-old rat). The results of the present study indicated that, when a relatively small number of granule cells are analyzed, one should take the heterogeneity of synaptic contacts on granule cells in number and type into consideration.     

The dendritic morphology of hippocampal dentate granule cells varies with their position in the granule cell layer: a quantitative Golgi study

Summary The dendritic morphology of Golgistained hippocampal dentate granule cells was evaluated by measuring the amount and location of dendrite, and the number of length of dendritic branches. Granule cells with somata in the superficial third of the granule cell layer had substantially more dendritic material than those with somata in the deep portions of the cell layer; this difference occurred throughout the extent of the molecular layer. Superficial cells also had different dendritic branching patterns and wider dendritic fields than did cells located in the deeper two-thirds of the granule cell layer. These results indicate that the position of neurons within the cell layer should be taken into account when quantifying the dendritic fields of dentate granule cells.     

Rhythmic expression of per1 in the dentate gyrus is suppressed by corticosterone: Implications for neurogenesis

The stimulating action of anti-depressant drugs on the number of mitotic cells on the dentate gyrus on the adult rat depends on an intact diurnal rhythm of corticosterone. This suggests that there may be a clock mechanism in the dentate gyrus which is sensitive to corticoids. This paper reports the diurnal expression the ‘clock’ gene per1 in the dentate gyrus, and how it is altered by clamping the diurnal rhythm in corticosterone. We show that there is a diurnal rhythm in the number of mitotic progenitor cells in the dentate gyrus of the hippocampus in adult male per1-luciferase rats, approximately 6 h out of phase with the plasma corticosterone rhythm. This is suppressed by clamping the daily corticosterone levels by a subcutaneous implant of corticosterone (100 mg). There was also a daily rhythm of per1 expression in both suprachiasmatic nucleus (SCN) and the dentate gyrus, which were in phase with one another. The per1 rhythm in the dentate gyrus, but not the SCN, was suppressed by clamping the plasma corticosterone rhythm. These results are related to the previous finding that clamping the corticosterone rhythm also prevents the stimulating action of fluoxetine and other controlling agents on the mitotic activity of the progenitor cells.     

Chronic treatment of exendin-4 affects cell proliferation and neuroblast differentiation in the adult mouse hippocampal dentate gyrus

Exendin-4 isolated from Heloderma suspectum venom acts via glucagon-like peptide 1 (GLP-1) receptor and has clinically been used in the type 2 diabetes. In this study, we investigated the effects of exendin-4 on cell proliferation and neuroblast differentiation in the subgranular zone (SGZ) of the dentate gyrus in mice. Exendin-4 was treated intraperitoneally to male ICR mice twice a day for 21 days. The exendin-4-treated group showed a significantly higher number of Ki67- (1.51-fold), doublecortin (DCX)- (2.5-fold) and 5-bromo-2′-deoxyuridine (BrdU) + DCX- (2.46-fold) immunoreactive cells in the SGZ of the dentate gyrus compared to the control group. The results of this study showed that treatment with exendin-4 increased cell proliferation neuroblast differentiation in the SGZ of the dentate gyrus, suggesting that exendin-4 promotes structural plasticity in the dentate gyrus.     

Changes in the total number of dentate granule cells in juvenile and adult rats: A correlated volumetric and 3H-thymidine autoradiographic study

Summary The total number of granule cells in the dentate gyrus was estimated in 17 male rats, four each aged 30, 120, and 200 days, and five aged 365 days. There is a substantial 35–43% linear increase between 1 month and 1 year. Two parameters of the granular layer are involved in the numerical change. First, total granular layer volume grows linearly with age. Second, average volume of a single granule cell nucleus in the ventral dentate gyrus decreases with age. Older rats tend to have a larger granular layer filled with more and smaller cells. In another group of 21 male rats, ³H-thymidine injections were given on four consecutive days during juvenile (30–33, n = 6) and adult life (60–63, n = 5; 120–123, n = 6; 180–183, n = 4). All animals survived to 200 days of age. The proportion of labeled mature granule cells and labeled presumptive granule cell precursors were determined in anatomically-matched slices. With older ages at injection, there is a decline in labeled mature granule cells and a concurrent increase in labeled precursors. These data are compatible with the constant level of granule cell increase determined volumetrically. Most of the late granule cells originate nearly simultaneously along the base of the main bulk of the granular layer; very few are found in the dorsal tip (septal extreme) and ventral tip (temporal extreme). This study is the first demonstration of a net numerical gain in a neuronal population during adulthood in the mammalian brain. Since the granule cells play a pivotal role in hippocampal function, these data suggest that their influence grows with age.Supported by the National Science Foundation (Grant No. BNS 79-21303)     

Visuospatial learning and memory in the Cebus apella and microglial morphology in the molecular layer of the dentate gyrus and CA1 lacunosum molecular layer

We investigated whether the morphology of microglia in the molecular layer of the dentate gyrus (DG-Mol) or in the lacunosum molecular layer of CA1 (CA1-LMol) was correlated with spatial learning and memory in the capuchin monkey (Cebus apella). Learning and memory was tested in 4 monkeys with visuo-spatial, paired associated learning (PAL) tasks from the Cambridge battery of neuropsychological tests. After testing, monkeys were sacrificed, and hippocampi were sectioned. We specifically immunolabeled microglia with an antibody against the adapter binding, ionized calcium protein. Microglia were selected from the middle and outer thirds of the DG-Mol (n = 268) and the CA1-LMol (n = 185) for three-dimensional reconstructions created with Neurolucida and Neuroexplorer software. Cluster and discriminant analyses, based on microglial morphometric parameters, identified two major morphological microglia phenotypes (types I and II) found in both the CA1-LMol and DG-Mol of all individuals. Compared to type II, type I microglia were significantly smaller, thinner, more tortuous and ramified, and less complex (lower fractal dimensions). PAL performance was both linearly and non-linearly correlated with type I microglial morphological features from the rostral and caudal DG-Mol, but not with microglia from the CA1-LMol. These differences in microglial morphology and correlations with PAL performance were consistent with previous proposals of hippocampal regional contributions for spatial learning and memory. Our results suggested that at least two morphological microglial phenotypes provided distinct physiological roles to learning-associated activity in the rostral and caudal DG-Mol of the monkey brain.     

Breaches of the pial basement membrane are associated with defective dentate gyrus development in mouse models of congenital muscular dystrophies

A subset of congenital muscular dystrophies (CMDs) has central nervous system manifestations. There are good mouse models for these CMDs that include POMGnT1 knockout, POMT2 knockout and Large^myd mice with all exhibiting defects in dentate gyrus. It is not known how the abnormal dentate gyrus is formed during the development. In this study, we conducted a detailed morphological examination of the dentate gyrus in adult and newborn POMGnT1 knockout, POMT2 knockout, and Large^myd mice by immunofluorescence staining and electron microscopic analyses. We observed that the pial basement membrane overlying the dentate gyrus was disrupted and there was ectopia of granule cell precursors through the breached pial basement membrane. Besides these, the knockout dentate gyrus exhibited reactive gliosis in these mouse models. Thus, breaches in the pial basement membrane are associated with defective dentate gyrus development in mouse models of congenital muscular dystrophies.     

Induction of cell proliferation and neuroblasts in the subgranular zone of the dentate gyrus by aqueous extract from Platycodon grandiflorum in middle-aged mice

In the present study, we observed the neurogenic effects of an aqueous extract from the root of Platycodon grandiflorum (EPG) in middle-aged (12-month-old) mice. For this, 100mg/kg EPG was administered orally to mice for 30 days before sacrifice and 5-bromodeoxyguanosine (BrdU) was injected intraperitoneally every 8h for 24h on the day prior to sacrifice. The increase of neurogenesis was estimated by immunohistochemical staining for cellular proliferation markers (BrdU and Ki67) and a marker for neuroblasts (Doublecortin, DCX). These markers were detected in the subgranular zone of the dentate gyrus in vehicle- and EPG-treated groups. The number of BrdU-, Ki67- and DCX-positive cells in the EPG-treated group was significantly increased compared to that in the vehicle-treated group. In addition, DCX-positive cells in the EPG-treated group showed well-developed processes. These results suggest that the number of neuroblasts is increased by the repeated treatment of EPG in middle-aged mice.     

1型糖尿病脑病大鼠空间学习记忆与海马齿状回神经干细胞增殖的观察

目的揭示1型糖尿病继发性脑病变(糖尿病脑病)的发生与海马齿状回颗粒下区(SGZ)神经干细胞增殖之间的关系。方法应用链脲佐菌素(溶解于柠檬酸缓冲液中)腹腔注射,将成年雄性Wistar大鼠建立成1型糖尿病脑病模型;将用柠檬酸缓冲液腹腔注射的大鼠或正常大鼠分别作为载体模型组或正常对照组。在建立模型成功后的60d,用Morris水迷宫和5-溴脱氧尿嘧啶(BrdU,一种神经干细胞合成DNA的标记物)免疫组化方法,观察各组大鼠空间学习记忆能力以及SGZ神经干细胞(BrdU阳性细胞)的变化。结果1型糖尿病脑病模型大鼠的逃避潜伏期和游泳距离均较载体模型组或正常对照组大鼠明显延长(P<0.01);而且该脑病组大鼠SGZ的BrdU阳性细胞数也明显低于载体模型组或正常对照组大鼠(P<0.01)。结论个体内长期缺乏胰岛素可导致SGZ神经干细胞增殖出现障碍,从而引发空间学习记忆能力下降,这可能是诱发1型糖尿病脑病的一个因素。     

Association analysis of the DISC1 gene with schizophrenia in the Japanese population and DISC1 immunoreactivity in the postmortem brain

The Disrupted-in-Schizophrenia 1 (DISC1) gene plays a role in the regulation of neural development. Previous evidence from genetic association and biological studies implicates the DISC1 gene as having a role in the pathophysiology of schizophrenia. In the present study, we explored the association between DISC1 missense mutation rs821616 (Ser704Cys) single nucleotide polymorphism (SNP) and four other SNPs (rs1772702, rs1754603, rs821621, rs821624) in the related haplotype block and schizophrenia in the Japanese population. We could not find a significant association of selected SNPs with schizophrenia after correction for multiple testing. We performed a meta-analysis of the Ser704Cys variant in schizophrenia using data from the present study and five previous Japanese population studies, and found no association with schizophrenia. We also examined DISC1 immunoreactivity in postmortem prefrontal cortex specimens of schizophrenia patients compared to control samples. The immunoreactivity revealed a significant decrease of DISC1 protein expression in the schizophrenia samples after ruling out potential confounding factors. However, the Ser704Cys variant did not show effects on DISC1 immunoreactivity. These results provide evidence that this functional genetic variation of DISC1 do not underlie the pathophysiology of schizophrenia in the Japanese population.     

Decreased expression of intercellular adhesion molecule-1 (ICAM-1) and urokinase-type plasminogen activator receptor (uPAR) is associated with tumor cell spreading in vivo

The development of an effective antitumor immune response to control tumor growth is influenced by the tumor cell itself and/or by the tumor microenvironment. Tumor invasion and tumor cell spreading require a finely tuned regulation of the formation and loosening of adhesive contacts of tumor cells with the extracellular matrix (ECM). In our laboratory, a rat tumor cell line derived from a spontaneous rat sarcoma revealed, by flow cytometry, a high frequency of intercellular adhesion molecule-1 (ICAM-1, 70.1 ± 8.7%) and urokinase-type plaminogen activator receptor (uPAR, 51.2 ± 5.2%) positive cells, while a weak expression of MHC class II (IA, 2.2±0.2% and IE, 17.4±3.7%) and B7 (12.1±2.2%) antigens was detected. In our tumor experimental model, after implantation of tumor cells, visible tumor masses were present at days 5–7 with a relatively fast tumor growth until day 15 (progressive phase) followed by a suppression of the tumor growth (regressive phase). Here we present data that correlates a significant decrease in the frequency of ICAM-1 and uPAR expressing tumor cells with the appearance of tumor cells in sites distant from that of the primary tumor. In addition we describe the development of a cellular immune response which controls the tumor progression and is associated with an increase in the expression of major histocompatibility complex (MHC) class II IA antigen during tumor development. The histological examination at tumor progressive and regressive time points revealed the relevant presence of polymorphonuclear neutrophils (PMNs) evidencing colliquative necrosis in tumor growth areas. Taken together, these results support the idea that the balance between adhesive interactions, proteolytic activity and tumorigenicity may lead to a tumor invasive phenotype. This revised version was published online in July 2006 with corrections to the Cover Date.     

Decreased expression levels of DAL-1 and TOB1 are associated with clinicopathological features and poor prognosis in gastric cancer

PurposeWe previously demonstrated that the functional inactivation of DAL-1 and TOB1 promotes an aggressive phenotype in gastric cancer cells, but the links between both genes and the survival of patients with gastric cancer are unknown. Here, we investigated the correlations of the expression levels of DAL-1 and TOB1 with the progression of gastric cancer.MethodsA total of 270 patients who underwent resectable gastrectomy were included. The expression of DAL-1 and TOB1 was detected by immunohistochemistry.ResultsLow expression of DAL-1 in cancer tissue was significantly associated with tumor site (p < 0.05), histological grade (p < 0.01), depth of invasion (p < 0.05), lymph node metastasis status (p < 0.05), Lauren classification (p < 0.001), and clinical stage (p < 0.01). A lower level of TOB1 was observed in gastric cancer patients with diffuse type disease compared to patients with either intestinal or mixed type disease (p < 0.001). Additionally, Spearman’s correlation analysis revealed that decreased expression of DAL-1 was positively correlated with low TOB1 expression (r=0.304, p < 0.001). The survival analysis showed that low levels of DAL-1 and TOB1 were significantly associated with poor survival of gastric cancer patients (p <0.001 and p < 0.05, respectively).ConclusionThe downregulation of DAL-1 and TOB1 expression is associated with shorter survival of gastric cancer patients. Hence, DAL-1 and TOB1 may be considered potential novel markers for predicting the outcomes of patients with gastric cancer.     

Effects of changes in extracellular potassium,magnesium and calcium concentration on synaptic transmission in area CA1 and the dentate gyrus of rat hippocampal slices

The dependence of stimulus-induced synaptic potentials on changes of extracellular ionic concentrations of potassium ([K⁺]_o 3, 5, 8 mM), magnesium ([Mg²⁺]_o 2, 4, 8 mM) and calcium [Ca²⁺]_o (2 mM and continuous lowering by washing with Ca²⁺-free solutions) was investigated in area CA1 and dentate gyrus of rat hippocampal slices. Field potentials (fps), [K⁺]_o and [Ca²⁺]_o were measured with double-barreled ion selective/reference microelectrodes. Paired pulse stimulation (interval 50-ms) was applied either to the lateral perforant path or to the Schaffer collaterals. Elevation of [K⁺]_o from 5 to 8 mM and of [Mg²⁺]_o from 2 to 8 mM depressed the rise of excitatory postsynaptic potentials, as well as the amplitude of population spikes. With elevation of [K⁺]_o, the effect was stronger in the dentate gyrus, while with elevation of [Mg²⁺]_o, the reduction was more pronounced in area CA1. During washout of Ca²⁺, synaptic potentials became reduced and finally depressed. The [Ca²⁺]_o at which synaptic transmission was blocked increased with higher [Mg²⁺]_o and decreased with a change of [K⁺]_o from 3 to 5 mM, whereas with an elevation of [K⁺]_o from 5 to 8 mM, it rose in area CA1 but was reduced in dentate gyrus. All ionic changes also affected frequency habituation and potentiation in paired pulse experimentes. In dentate gyrus, frequency habituation was reversed to frequency potentiation with moderate lowering of [Ca²⁺]_o and with elevation of [Mg²⁺]_o and [K⁺]_o. In contrast, in area CA1 frequency potentiation was reduced upon elevation of [K⁺]_o.     

Mutations in the planar cell polarity genes CELSR1 and SCRIB are associated with the severe neural tube defect craniorachischisis

Craniorachischisis (CRN) is a severe neural tube defect (NTD) resulting from failure to initiate closure, leaving the hindbrain and spinal neural tube entirely open. Clues to the genetic basis of this condition come from several mouse models, which harbor mutations in core members of the planar cell polarity (PCP) signaling pathway. Previous studies of humans with CRN failed to identify mutations in the core PCP genes, VANGL1 and VANGL2. Here, we analyzed other key PCP genes: CELSR1, PRICKLE1, PTK7, and SCRIB, with the finding of eight potentially causative mutations in both CELSR1 and SCRIB. Functional effects of these unique or rare human variants were evaluated using known protein-protein interactions as well as subcellular protein localization. While protein interactions were not affected, variants from five of the 36 patients exhibited a profound alteration in subcellular protein localization, with diminution or abolition of trafficking to the plasma membrane. Comparable effects were seen in the crash and spin cycle mouse Celsr1 mutants, and the line-90 mouse Scrib mutant. We conclude that missense variants in CELSR1 and SCRIB may represent a cause of CRN in humans, as in mice, with defective PCP protein trafficking to the plasma membrane a likely pathogenic mechanism.     

Clioquinol and vitamin B12 (cobalamin) synergistically rescue the lead-induced impairments of synaptic plasticity in hippocampal dentate gyrus area of the anesthetized rats in vivo

Lead (Pb(2+)) exposure in development induces impairments of synaptic plasticity in the hippocampal dentate gyrus (DG) area of the anesthetized rats in vivo. The common chelating agents have many adverse effects and are incapable of alleviating lead-induced neurotoxicity. Recently, CQ, clioquinol (5-chloro-7-iodo-8-hydroxy-quinoline), which is a transition metal ion chelator and/or ionophore with low affinity for metal ions, has yielded some promising results in animal models and clinical trials related to dysfunctions of metal ions. In addition, CQ-associated side effects are believed to be overcome with vitamin B12 (VB12) supplementation. To determine whether CQ treatment could rescue impairments of synaptic plasticity induced by chronic Pb(2+) exposure, we investigated the input/output functions (I/Os), paired-pulse reactions (PPRs) and long-term potentiation (LTP) of different treatment groups in hippocampal DG area of the anesthetized rat in vivo by recording field potentials and measured hippocampal Pb(2+) concentrations of different treatment groups by PlasmaQuad 3 inductive coupled plasma mass spectroscopy. The results show: CQ alone does not rescue the lead-induced impairments of synaptic plasticity in hippocampal DG area of the anesthetized rats in vivo; VB12 alone partly rescues the lead-induced impairments of LTP; however the co-administration of CQ and VB12 totally rescues these impairments of synaptic plasticity and moreover, the effects of CQ and VB12 co-administration are specific to the lead-exposed animals.     

Expression of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) in normal human Sertoli cells and its up-regulation in impaired spermatogenesis

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is usually expressed at the luminal surface of different epithelia and is up-regulated in endothelial cells during angiogenesis. Here, we demonstrate evidence of morphogenetic effects of CEACAM1 in spermatogenesis. CEACAM1 is detectable in normal testicular tissue and seminal fluid. It is present in the adluminal part of Sertoli cells extending only as far as the tight junctions between them. CEACAM1 immunostaining is significantly increased and extends to the basal part of Sertoli cells in the presence of carcinoma in situ. Also, in vitro-induced spermatogenetic disturbance leads to an enhanced CEACAM1 expression in Sertoli cells after 3 days of culture. Remarkably, seminiferous tubules containing exclusively Sertoli cells do not exhibit any CEACAM1 expression. CEACAM1 staining was absent in vascular endothelial cells of normal testicular tissue, but present in small blood vessels of seminomas. These data suggest that CEACAM1 expression in Sertoli cells depends on the presence of germ cells and plays a role in adhesive interactions between Sertoli and differentiating germ cells. Its up-regulation in Sertoli cells accompanying spermatogenic damage may contribute to reconstruction and maintenance of the tubular structure of seminiferous tubules. Additionally, CEACAM1 is apparently involved in the angiogenesis of germ cell tumours.     

Decreased Beclin-1 expression is correlated with the growth of the primary tumor in patients with squamous cell carcinoma and adenocarcinoma of the lung

Beclin-1 is a Bcl-2-interacting protein, and it may delay cell cycle progression and induce autophagy. The function and expression of Beclin-1 and Bcl-2 in squamous cell carcinoma and adenocarcinoma of the lung remain largely unknown. Herein, we investigated the expression of Beclin-1 and Bcl-2 in squamous cell carcinoma and adenocarcinoma of the lung. Tissue samples from 262 cases were used in this study. Immunohistochemical staining for Beclin-1 and Bcl-2 were conducted using a tissue microarray. In squamous cell carcinoma, Beclin-1 expression was strongly positive in 48 (28.6%) of 168 samples, it was moderately positive in 42 (25.0%) of 168 samples, and it was negative or weakly positive in 78 (46.4%) of 168 samples. In adenocarcinoma, Beclin-1 expression was strongly positive in 26 (27.7%) of 94 samples, it was moderately positive in 27 (28.7%) of 94 samples, and it was negative or weakly positive in 41 (43.6%) of 94 samples. Beclin-1 expression was inversely correlated with the tumor size and the primary tumor stage (pT) in both types of tumor. Especially, the TNM stage of adenocarcinoma was inversely correlated with Beclin-1 expression. Our results suggest that a progressively reduced Beclin-1 expression is correlated with the primary tumor growth of squamous cell carcinoma and adenocarcinoma of the lung.     

Expression of the EphA1 protein is associated with Fuhrman nuclear grade in clear cell renal cell carcinomas

Aberrant expression of receptor tyrosine kinase EphA1 in malignant tissues has been reported. However, the expression profile of EphA1 in renal cell carcinoma (RCC) and its association with clinicopathological parameters remain unknown. The aim of this study was to determine the cancerous value of the EphA1 protein expression in patients with renal cell carcinomas. This study included 144 patients with clear cell RCC (ccRCC), 18 patients with chromophobe RCC and 6 patients with papillary RCC. The EphA1 protein was detected in RCC tissue samples by an immunohistochemical staining with a specific polycolonal antibody. The correlation of the expression of the EphA1 protein with clinicopathological parameters was evaluated. High level of the expression of EphA1 was observed in all normal renal tubes. The EphA1 protein was negatively or weakly expressed in 93 out of 144 ccRCC (64.6%) and positively expressed in 51 out of 144 ccRCC (35.4%). The high level expression of the EphA1 protein was significantly associated with younger patients (P<0.001), sex (P=0.016) and lower nuclear grade (P<0.001). No significant relation between the expression of EphA1 and tumor diameter was found (P=0.316). Positive expression of EphA1 was observed in all samples of chromophobe RCC and papillary RCC. Our data indicated that the EphA1 protein may be a new marker for the prognosis of ccRCC.