Gene expression in developing rat hippocampal pyramidal neurons appears independent of mossy fiber innervation.

In the rat, neonatal gamma-irradiation of the hippocampus induces a selective destruction of dentate granule cells and prevents the development of the mossy fiber-CA3 pyramidal cell connection. In the absence of mossy fiber input, the CA3 pyramidal neurons exhibit morphological alterations and rats deprived of dentate granule cells fail to develop kainate-induced epileptic activity in the CA3 pyramidal neurons. Neonatal elimination of the granule cells also impairs learning and memory tasks in adult rats. In the present work, we assessed by in situ hybridization and semi-quantitative RT-PCR, whether in the pyramidal layers, the absence of mossy fiber input alters the expression of a number of genes involved in activity-dependent signal transduction, in GABAergic neurotransmitter signaling and in neurite development via microtubule organization. Surprisingly, we show that the expression and the developmentally regulated alternative splicing of the genes we examined in the developing hippocampus are not altered in the pyramidal neurons, whether the dentate granule afferents are present or absent. Our results suggest that in the CA3 pyramidal layer, the developmental expression patterns of the mRNAs we studied are independent of extrinsic cues provided by mossy fiber input.     

Gas7在大鼠海马和齿状回发育过程中的动态表达

目的研究生长休止蛋白7（Gas7）在大鼠海马和齿状回不同发育阶段的表达。方法采用免疫组织化学方法观察Gas7在SD大鼠胚胎第18d（E18）、新生（P0）、生后第7d（P7）、P14、P21和成年海马和齿状回中的表达和分布。结果在大鼠脑海马和齿状回部位的冠状切片上,Gas7免疫反应阳性产物主要表达在海马的锥体细胞、齿状回的颗粒细胞和门区的多形层细胞。随着发育的进程,在海马,Gas7较早表达在CA3区,其次是CA2和CA1区;在齿状回,Gas7在外臂的表达早于内臂,在颗粒细胞层的表达是按先外层后内层的顺序。在围生期,Gas7在海马和齿状回各区的表达逐渐增强,至P14达到高峰,后逐渐降低,至P21其表达强度和分布趋于恒定至成年水平。结论 Gas7在大鼠海马和齿状回发育过程中的动态表达具有时间和空间上的特异性,提示Gas7可能参与了海马和齿状回形态形成和功能成熟的调控。     

In vivo neuroprotective role of NMDA receptors against kainate-induced excitotoxicity in murine hippocampal pyramidal neurons

Activation of NMDA receptors has been shown to induce either neuronal cell death or neuroprotection against excitotoxicity in cultured cerebellar granule neurons in vitro. We have investigated the effects of pretreatment with NMDA on kainate-induced neuronal cell death in mouse hippocampus in vivo. The systemic administration of kainate (30 mg/kg), but not NMDA (100 mg/kg), induced severe damage in pyramidal neurons of the hippocampal CA1 and CA3 subfields 3-7 days later, without affecting granule neurons in the dentate gyrus. An immunohistochemical study using an anti-single-stranded DNA antibody and TdT-mediated dUTP nick end labeling analysis both revealed that kainate, but not NMDA, induced DNA fragmentation in the CA1 and CA3 pyramidal neurons 1-3 days after administration. Kainate-induced neuronal loss was completely prevented by the systemic administration of NMDA (100 mg/kg) 1 h to 1 day previously. No pyramidal neuron was seen with fragmented DNA in the hippocampus of animals injected with kainate 1 day after NMDA treatment. The neuroprotection mediated by NMDA was prevented by the non-competitive NMDA receptor antagonist MK-801. Taken together these results indicate that in vivo activation of NMDA receptors is capable of protecting against kainate-induced neuronal damage through blockade of DNA fragmentation in murine hippocampus.     

小鼠海马内HDAC2的表达及其与NMDA受体、PSD-95的共定位

目的探讨组蛋白去乙酰化酶2（HDAC2）在成年C57BL/6小鼠海马内的分布及其与突触后致密区（PSD）蛋白成员的共定位,为揭示HDAC2与PSD蛋白复合物之间的内在联系及在海马相关的学习记忆过程中可能起到的调控作用提供形态学依据。方法应用免疫组化方法观察HDAC2在C57BL/6小鼠海马各区的表达分布。应用免疫荧光双标技术研究HDAC2与PSD蛋白成员N-甲基-D-天冬氨酸（NMDA）受体亚单位1（NR1）、PSD-95之间是否存在共定位。结果 HDAC2在小鼠海马CA1～CA3区锥体细胞和齿状回颗粒细胞均具有明显表达,而在各区的始层、辐射层、腔隙-分子层以及齿状回多形细胞层表达均较少。免疫荧光双标染色图片的重叠表明,HDAC2与NR1、PSD-95在小鼠海马CA1～CA3区锥体细胞层和齿状回颗粒细胞层内均可见显著共表达现象,其他区域偶见散在分布的双染神经元。结论 HDAC2在小鼠海马锥体细胞层和颗粒细胞层表达丰富,并与PSD蛋白成员间存在共定位现象。本实验结果为探讨HDAC2对谷氨酸能突触后神经元依赖的突触可塑性的调节机制提供了形态学依据。     

Cre-mediated cerebellum- and hippocampus-restricted gene mutation in mouse brain

Using the phage P1-derived Cre/loxP recombination system, we have created a line of cre-transgenic mice in which the Cre-mediated gene deletion is restricted to granule cells of cerebellum and dentate gyrus of hippocampus. Low levels of deletion were also present in pyramidal cells of hippocampal CA1 and CA3 fields. The Cre/loxP recombination occurred prenatally. The recombination efficiencies in the granular layer of the cerebellum, the granular layer of the dentate gyrus, and the CA1 and CA3 pyramidal cells of the hippocampus were 34.0%, 23.1%, 3.0%, and 9.8%, respectively. This line of cre-transgenic mice should be conducive to studies of the effect of a gene mutation upon brain development and plasticity.     

Comparative mapping of opioid receptors and enkephalin immunoreactive nerve terminals in the rat hippocampus. A radiohistochemical and immunocytochemical study

Opioid receptors can be localized to the hippocampal formation of the rat by autoradiography. The binding of 3H-enkephalinamide to fixed and mounted tissue sections has all the characteristics associated with binding to opioid receptors. It is saturable, of high affinity and displays stereospecificity. The opioid receptor distribution shows striking regional variation throughout the hippocampal formation. Areas with high density include the pyramidal cell layer of both regio superior (CA1) and regio inferior (CA3), stratum moleculare of the hippocampus, the cell layer of subiculum, the superficial part of presubiculum and the deep layer (VI) of the medial and lateral entorhinal cortices. Areas with low to medium densities include regions corresponding to the dendritic field of the pyramidal cells (str. oriens, str. radiatum and the mossy fiber zone), the dentate granule cell layer and the molecular layer of the dentate area. Enkephalin-like immunoreactivity is detected in both intrinsic neuronal systems: 1) the mossy fibers which terminate on the proximal part of the CA3 pyramidal cell dendrites and on CA4 pyramidal cells, 2) cell bodies with multiple short processes, probably interneurons, dispersed throughout the hilus of the dentate area, the pyramidal cell layer of hippocampus, the str. radiatum, and occasionally in the str. moleculare and in the str. oriens, and extrinsic neuronal systems: 1) the lateral perforant path and 2) the lateral temporo-ammonic tract. Thus, the hippocampus contains intrinsic systems of enkephalin-like immunoreactive nerve terminals which may exert their effect on the opioid receptors with a localization corresponding to the pyramidal cells and their apical dendrites. Extrinsic enkephalinergic systems corresponding to the terminal fields of the lateral perforant path and the temporoammonic tract, both of entorhinal origin, may influence the opioid receptors located in the molecular layer of the dentate area, and in the molecular layer of the hippocampus and the subiculum. Thus, the enkephalin-like immunoreactive nerve terminals are all located in areas which contain opioid binding sites. This suggests that the "opioid peptide-opioid receptor" systems may regulate hippocampal neuronal activity via neurotransmission or neuromodulation. However, a high or medium number of opioid binding sites occur over the pyramidal cell bodies and the dentate granule cell bodies, and these opioid binding sites are not in close contact with the major enkephalinergic systems.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neuroprotection with caspase-9 inhbition against in vitro and in vivo trauma

Previous work has suggested that a major contributor to neuronal cell death is the aberrant induction of the cell cycle process, as indicated by an up-regulation of cyclin D. In order to examine the temporal and spatial relationship of cyclin D in a model of acute neurodegeneration, the hippocampal toxicant, trimethyltin (TMT; 2.0 mg/kg), was administered to 21-day old CD−1 male mice and the level and cellular localization of cyclin D1 examined. Within 24 h following TMT, dentate granule cells of the hippocampus showed evidence of neuronal necrosis resulting in severe cell loss over a 3-day period. The pyramidal cell layer was spared with only sparse punctate neuronal necrosis. Microglia response was seen at 72 h with ameboid microglia present in the dentate and ramified microglia present in the pyramidal cell layer, contributing to the elevation seen in TNF-alpha mRNA levels. A transient elevation was seen in mRNA levels for cyclin D1 over 48–72 h post-TMT. Immunohistochemistry demonstrated a transient increase in staining for cyclin D1 in CA1 pyramidal neurons as early as 24 h. Punctate staining occurred in neurons throughout the dentate at 48 h. BrdU positive cells were present along the inner blades of the dentate in control animals. Following TMT exposure, an increase was seen in both the number of neurons stained and a diffusion of the staining pattern into the full dentate region. Thus, in TMT-induced neurodegeneration, cyclin D1 is not expressed in the vulnerable neurons but rather in neurons spared from degeneration. This expression pattern appears to not be linked to an increase in the cellular processes for proliferation as the majority of BrdU positive cells were present in the region of neuronal damage.     

Caspase-3 immunoreactivity in different cortical areas of young and aging macaque (Macaca mulatta) monkeys

It has been shown that cytochrome-c-dependent caspase-3 activation is significantly elevated in the aging macaque brain. To assess the underlying age-related changes in the cellular distribution of caspase-3, we have examined the motor cortex, cerebellum and hippocampus of young (4-year-old, n = 4) and old (20-year-old, n = 4)rhesus monkeys by immunohistochemistry. Western blot analyses of brain homogenate showed that the antibody reacted only with inactive 32-kDa procaspase and its active 20- and 17-kDa subunits, formed after granzyme B exposure. In the motor cortex, pyramidal cells of layers III and V were moderately labeled; the underlying white matter contained weakly stained astrocytes. In the hippocampus, hilar neurons and pyramidal cells in CA3 showed the strongest immunoreaction, pyramidal cells in CA1 and granule cells of the dentate gyrus were also strongly labeled. In contrast, CA2 pyramidal cells were only weakly stained, and neurons of the molecular layer were unlabeled. Weak caspase-3 immunoreaction of CA2 neurons parallels known decreased susceptibility to apoptosis. In the cerebellar cortex, clusters of strongly labeled Purkinje cells were observed next to groups of weakly and unstained cells; granule cells were generally unstained. The brains of aging monkeys displayed a similar pattern of caspase-3 immunoreactivity. In neocortical layer V, however, scattered very strongly labeled pyramidal cells were regularly detected, which were not observed in younger animals. This clustering of caspase-3 indicates increased vulnerability of a subset of pyramidal cells in the aging brain.     

Localization of type I insulin-like growth factor receptor messenger RNA in the adult rat brain by in situ hybridization.

Using multiple 35S-labeled oligonucleotide probes concurrently, the type I insulin-like growth factor receptor (IGF-I-R) mRNA was demonstrated by Northern blot hybridization in newborn and adult rat brain as a single species of approximately 11 kilobases. The probes were used to localize IGF-I-R mRNA by in situ hybridization in slices of adult rat brain. The highest levels of IGF-I-R mRNA expression were found in the glomerular and mitral cell body layers of the olfactory bulb, the granule cell body layers of the dentate gyrus and cerebellum, the pyramidal cell body layers of the piriform cortex and Ammon's horn, and the choroid plexus. The lowest levels of IGF-I-R mRNA expression were found in white matter. At the cellular level, IGF-I-R mRNA was expressed by a variety of neurons, by epithelial cells of the choroid plexus, and by ependymal cells of the third ventricle. Of the neuron types studied, the highest levels of IGF-I-R mRNA were consistently found in perikarya of mitral and tufted cells in the olfactory bulb, in pyramidal cells of the piriform cortex and Ammon's horn, and in granule cells of the dentate gyrus. There was a close congruency between the distribution of IGF-I binding and IGF-I-R mRNA at the regional level. Neuropil layers in the cerebral cortex, olfactory bulb, hippocampus, and cerebellum contained a high level of IGF-I binding, whereas the adjacent cell body layers contained a high level of the IGF-I-R mRNA. We conclude that in these regions, IGF-I-R mRNA is synthesized in neuronal cell bodies, and the receptors are transported to axons and dendrites in adjacent synapse-rich layers, where appropriate IGF effects are achieved.     

Expression of fatty acid-CoA ligase 4 during development and in brain

Fatty acid utilization is initiated by fatty acid-CoA ligase, which converts free fatty acids into fatty acyl-CoA esters. We have cloned previously the human long-chain fatty acid-CoA ligase 4 (FACL4), which is a central enzyme in controlling the free arachidonic acid level in cells and thereby regulating eicosanoid production. We report here the expression of this gene in tissues, particularly in different parts of the brain. We found that FACL4 encoded a 75 kDa enzyme and that there was a modified translation product expressed in the brain. FACL4 was expressed in early stages of development with a significant amount of FACL4 mRNA detected in an E7 mouse embryo. In addition, FACL4 was highly expressed in both adult and newborn mouse brain especially in the granule cells of the dentate gyrus and the pyramidal cell layer of CA1 in hippocampus, and the granular cell layer and Purkinje cells of the cerebellum.     

牦牛海马的形态特征及成体神经发生的初探

采用传统H.E 染色和Golgi-Cox 染色方法观察成年牦牛海马结构的形态和细胞构筑,并通过DCX - DAB免疫组化染色和DCX/ NeuN、GFAP / NeuN 双重免疫荧光标记等技术观察齿状回颗粒下层中的新生神经元和放射状胶质细胞。结果表明,牦牛海马结构主要包括齿状回和海马本部,二者分层清晰。海马的主要细胞为颗粒细胞、苔藓细胞和锥体细胞。CA3 区的锥体细胞胞体较CA1 区的大,但其顶树突的平均长度较短。CA1 区的锥体细胞明显分为两层,而CA3 区的则为一层。DCX 阳性细胞的胞体主要集中在齿状回颗粒下层靠近门区处,沿颗粒层内侧单个或少数聚集分布。沿齿状回颗粒下层分布着一层GFAP 阳性的放射状胶质细胞样细胞,其胞质和单极性的细长突起均呈GFAP 阳性,而胞核为阴性。在整个海马结构中均有大量星形GFAP 阳性细胞散在分布,特别是海马分子层和门区内靠近颗粒层部分的密度较其它部位大。牦牛海马的形态结构与绵羊的相似,而与大鼠、小鼠、家猫、兔子等小型哺乳动物有一定差别。两种DCX 免疫组化实验结果表明在牦牛海马中存在着新生神经元。GFAP 免疫荧光标记表明,牦牛海马结构中分布有星形胶质细胞;特别是放射状胶质细胞。     

Transient increase in Zn2+ in hippocampal CA1 pyramidal neurons causes reversible memory deficit

The translocation of synaptic Zn(2+) to the cytosolic compartment has been studied to understand Zn(2+) neurotoxicity in neurological diseases. However, it is unknown whether the moderate increase in Zn(2+) in the cytosolic compartment affects memory processing in the hippocampus. In the present study, the moderate increase in cytosolic Zn(2+) in the hippocampus was induced with clioquinol (CQ), a zinc ionophore. Zn(2+) delivery by Zn-CQ transiently attenuated CA1 long-term potentiation (LTP) in hippocampal slices prepared 2 h after i.p. injection of Zn-CQ into rats, when intracellular Zn(2+) levels was transiently increased in the CA1 pyramidal cell layer, followed by object recognition memory deficit. Object recognition memory was transiently impaired 30 min after injection of ZnCl(2) into the CA1, but not after injection into the dentate gyrus that did not significantly increase intracellular Zn(2+) in the granule cell layer of the dentate gyrus. Object recognition memory deficit may be linked to the preferential increase in Zn(2+) and/or the preferential vulnerability to Zn(2+) in CA1 pyramidal neurons. In the case of the cytosolic increase in endogenous Zn(2+) in the CA1 induced by 100 mM KCl, furthermore, object recognition memory was also transiently impaired, while ameliorated by co-injection of CaEDTA to block the increase in cytosolic Zn(2+). The present study indicates that the transient increase in cytosolic Zn(2+) in CA1 pyramidal neurons reversibly impairs object recognition memory.     

Expression and Localization of Type II Diacylglycerol Kinase Isozymes δ and η in the Developing Mouse Brain

The functions of type II diacylglycerol kinase (DGK) δ and -η in the brain are still unclear. As a first step, we investigated the spatial and temporal expression of DGKδ and -η in the brains of mice. DGKδ2, but not DGKδ1, was highly expressed in layers II–VI of the cerebral cortex; CA–CA3 regions and dentate gyrus of hippocampus; mitral cell, glomerular and granule cell layers of the olfactory bulb; and the granule cell layer in the cerebellum in 1- to 32-week-old mice. DGKδ2 was expressed just after birth, and its expression levels dramatically increased from weeks 1 to 4. A substantial amount of DGKη (η1/η2) was detected in layers II–VI of the cerebral cortex, CA1 and CA2 regions and dentate gyrus of the hippocampus, mitral cell and glomerular layers of the olfactory bulb, and Purkinje cells in the cerebellum of 1- to 32-week-old mice. DGKη2 expression reached maximum levels at P5 and decreased by 4 weeks, whereas DGKη1 increased over the same time frame. These results indicate that the expression patterns of DGK isozymes differ from each other and also from other isozymes, and this suggests that DGKδ and -η play distinct and specific roles in the brain.     

Changes of hippocampal neurons after perinatal exposure to ethanol

The effect of ethanol on the structural development of the central nervous system was studied in offspring of Wistar rats, drinking 20 % ethanol during pregnancy and till the 28th day of their postnatal life. The structural changes in the hippocampus and dentate gyrus were analyzed at the age of 18, 35 and 90 days. A lower width of pyramidal and granular cell layers, cell extinction and fragmentation of numerous nuclei were found in all experimental animals compared to control animals. The extent of neural cell loss was similar in all monitored areas and in all age groups. At the age of 18 and 35 days, the degenerating cells were observed in the CA1 and CA3 area of the hippocampus and in the ventral and dorsal blade of the dentate gyrus. Numerous glial cells replaced the neuronal population of this region. Some degenerating cells with fragmented nuclei were observed at the age of 90 days. Our experiments confirmed the vulnerability of the developing central nervous system by ethanol intake during the perinatal period and revealed a long-lasting degeneration process in the hippocampus and dentate gyrus.     

mGluRs Modulate Strength and Timing of Excitatory Transmission in Hippocampal Area CA3

Excitatory transmission within hippocampal area CA3 stems from three major glutamatergic pathways: the perforant path formed by axons of layer II stellate cells in the entorhinal cortex, the mossy fiber axons originating from the dentate gyrus granule cells, and the recurrent axon collaterals of CA3 pyramidal cells. The synaptic communication of each of these pathways is modulated by metabotropic glutamate receptors that fine-tune the signal by affecting both the timing and strength of the connection. Within area CA3 of the hippocampus, group I mGluRs (mGluR1 and mGluR5) are expressed postsynaptically, whereas group II (mGluR2 and mGluR3) and III mGluRs (mGluR4, mGluR7, and mGluR8) are expressed presynaptically. Receptors from each group have been demonstrated to be required for different forms of pre- and postsynaptic long-term plasticity and also have been implicated in regulating short-term plasticity. A recent observation has demonstrated that a presynaptically expressed mGluR can affect the timing of action potentials elicited in the postsynaptic target. Interestingly, mGluRs can be distributed in a target-specific manner, such that synaptic input from one presynaptic neuron can be modulated by different receptors at each of its postsynaptic targets. Consequently, mGluRs provide a mechanism for synaptic specialization of glutamatergic transmission in the hippocampus. This review will highlight the variability in mGluR modulation of excitatory transmission within area CA3 with an emphasis on how these receptors contribute to the strength and timing of network activity within pyramidal cells and interneurons.