Epigenetic mechanisms in the dentate gyrus act as a molecular switch in hippocampus-associated memory formation

We make strong memories of significant events in our lives which may serve to increase our resilience and adaptation capacity to deal with future challenges. It is well established that the neurotransmitter glutamate and the ERK MAPK intracellular signaling pathway play a principal role in memory formation. In addition, stress-associated hormones like glucocorticoids released during such events are known to strengthen formation of memories. But, how do these hormones work? Do they interact with the ERK MAPK pathway or otherwise? What are the more distal, epigenomic effects? We discovered in rats and mice that confrontation with a psychological challenge (e.g. forced swimming, Morris water maze) would lead, through NMDA-ERK signaling, to MSK1 and Elk-1 activation in dentate gyrus neurons (a part of the hippocampus involved in encoding of memories) resulting in histone H3 S10-phosphorylation and K14-acetylation, H4 hyper-acetylation, gene induction and formation of memories of the event. Moreover, glucocorticoid hormones via the glucocorticoid receptor (GR) greatly facilitated the epigenomic mechanisms and cognitive performance. Therefore, we propose that formation of enduring memories of significant events requires an interaction of GRs with the NMDA/ERK/MSK1/Elk-1 signaling pathways to allow an optimal epigenomic activation pattern in dentate gyrus neurons to accommodate their altered neurophysiological function.     

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。     

Brain mineralocorticoid receptor diversity: Functional implications

Mineralocorticoid receptors (MRs) in neurons of the anterior hypothalamus and the periventricular brain regions mediate aldosterone-selective actions on sodium hemeostasis, salt appetite and cardiovascular regulation. Corticosterone is not effective in these neurons, possibly because it is enzymatically inactivated. However, MRs in limbic brain regions, notably in the hippocampal neurons, do already respond to very low concentrations of both corticosterone and aldosterone. The MR-mediated effects stabilize neuronal transmission and appear critical for neuronal integrity of a sub-region of the hippocampus: the dentate gyrus. Higher concentrations of corticosterone induced by stress and the circadian rise progressively activate the lower affinity glucocorticoid receptors (GRs), which in coordination with MR-mediated actions then facilitate adaptive processes required for recovery of homeostasis. It is postulated that this balanced MR- and GR-mediated action of corticosterone is of critical importance for regulation of the stress response and behavioural adaptation.     

Temporal Changes in Prosaposin Expression in the Rat Dentate Gyrus after Birth

Neurogenesis in the hippocampal dentate gyrus occurs constitutively throughout postnatal life. Adult neurogenesis includes a multistep process that ends with the formation of a postmitotic and functionally integrated new neuron. During adult neurogenesis, various markers are expressed, including GFAP, nestin, Pax6, polysialic acid-neural cell adhesion molecule (PSA-NCAM), neuronal nuclei (NeuN), doublecortin, TUC-4, Tuj-1, and calretinin. Prosaposin is the precursor of saposins A–D; it is found in various organs and can be excreted. Strong prosaposin expression has been demonstrated in the developing brain including the hippocampus, and its neurotrophic activity has been proposed. This study investigated changes in prosaposin in the dentate gyrus of young and adult rats using double immunohistochemistry with antibodies to prosaposin, PSA-NCAM, and NeuN. Prosaposin immunoreactivity was intense in the dentate gyrus at postnatal day 3 (P3) and P7, but decreased gradually after P14. In the dentate gyrus at P28, immature PSA-NCAM-positive neurons localized exclusively in the subgranular zone were prosaposin-negative, whereas mature Neu-N-positive neurons were positive for prosaposin. Furthermore, these prosaposin-negative immature neurons were saposin B-positive, suggesting that the neurons take up and degrade prosaposin. In situ hybridization assays showed that prosaposin in the adult dentate gyrus is dominantly the Pro+9 type, a secreted type of prosaposin. These results imply that prosaposin secreted from mature neurons stimulates proliferation and maturation of immature neurons in the dentate gyrus.     

Monosynaptic GABAergic signaling from dentate to CA3 with a pharmacological and physiological profile typical of mossy fiber synapses

Mossy fibers are the sole excitatory projection from dentate gyrus granule cells to the hippocampus, where they release glutamate, dynorphin, and zinc. In addition, mossy fiber terminals show intense immunoreactivity for the inhibitory neurotransmitter GABA. Fast inhibitory transmission at mossy fiber synapses, however, has not previously been reported. Here, we show that electrical or chemical stimuli that recruit dentate granule cells elicit monosynaptic GABA(A) receptor-mediated synaptic signals in CA3 pyramidal neurons. These inhibitory signals satisfy the criteria that distinguish mossy fiber-CA3 synapses: high sensitivity to metabotropic glutamate receptor agonists, facilitation during repetitive stimulation, and NMDA receptor-independent long-term potentiation. GABAergic transmission from the dentate gyrus to CA3 has major implications not only for information flow into the hippocampus but also for developmental and pathological processes involving the hippocampus.     

Differential expression of immediate early genes in the hippocampus and spinal cord

We have demonstrated that immediate early genes can be differentially activated within the central nervous system. We examined the effects of tetanic stimulation in the hippocampus and of noxious sensory stimulation of the spinal cord on the expression of eight immediate early genes. Induction of long-term potentiation (LTP) in the dentate gyrus resulted in an increase in mRNA and protein for NGFI-A (also termed Zif/268, Egr-1, or Krox 24), and less consistently for jun-B mRNA. No increase was seen for c-fos, NGFI-B, c-jun, jun-D, SRF, or PC4 mRNAs. Blockade of the NMDA receptor prevented the induction of both LTP and NGFI-A mRNA in the dentate gyrus. However, commissural stimulation, which prevented the induction of LTP, resulted in bilateral activation of all the genes examined, including NGFI-A. No change was seen in animals trained in a water maze. These results suggest that no simple relationship exists between LTP, spatial learning, and immediate early gene induction. Stimulation of sensory fibers resulted in an increase in mRNA for NGFI-A, c-fos, SRF, NGFI-B, and c-jun in spinal cord neurons. Blockade of the NMDA receptor had no effect on immediate early gene induction in the spinal cord.     

Excitotoxic damage of the hippocampus in the rat: involvement of N-methyl-D-aspartate receptors

The intrahippocampal injection of two agonists of excitatory aminoacid (EAA) receptors elicited neuronal damages localized in CA1 and dentate gyrus for N-methyl-D-aspartate (NMDA) (20 nmol) and extended to the various hippocampal areas, except dentate gyrus for kainic acid (KA) (2.5 nmol). The pretreatment of the animals with N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) (20 mg/kg), a noncompetitive NMDA-receptor antagonist, prevented the neuronal injury induced by NMDA and KA in CA1. The distribution of neuronal damages and of TCP-protected areas closely correlated to that of EEA-receptors and of TCP binding sites in the hippocampus.     

Neuroprotective effects of quercetin, rutin and okra (Abelmoschus esculentus Linn.) in dexamethasone-treated mice

The administration of dexamethasone, a synthetic glucocorticoid receptor agonist, causes neuronal death in the CA3 layer of the hippocampus, which has been associated with learning and memory impairments. This study aimed to examine the ability of okra (Abelmoschus esculentus Linn.) extract and its derivatives (quercetin and rutin) to protect neuronal function and improve learning and memory deficits in mice subjected to dexamethasone treatment. Learning and memory functions in mice were examined using the Morris water maze test. The results showed that the mice treated with dexamethasone had prolonged water maze performance latencies and shorter time spent in the target quadrant while mice pretreated with quercetin, rutin or okra extract prior to dexamethasone treatment showed shorter latencies and longer time spent in target quadrant. Morphological changes in pyramidal neurons were observed in the dexamethasone treated group. The number of CA3 hippocampal neurons was significantly lower while pretreated with quercetin, rutin or okra attenuated this change. Prolonged treatment with dexamethasone altered NMDA receptor expression in the hippocampus. Pretreatment with quercetin, rutin or okra extract prevented the reduction in NMDA receptor expression. Dentate gyrus (DG) cell proliferation was examined using the 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry technique. The number of BrdU-immunopositive cells was significantly reduced in dexamethasone-treated mice compared to control mice. Pretreatment with okra extract, either quercetin or rutin was found to restore BrdU-immunoreactivity in the dentate gyrus. These findings suggest that quercetin, rutin and okra extract treatments reversed cognitive deficits, including impaired dentate gyrus (DG) cell proliferation, and protected against morphological changes in the CA3 region in dexamethasone-treated mice. The precise mechanism of the neuroprotective effect of these plant extracts should be further investigated.     

慢性束缚应激大鼠中枢糖皮质激素受体变化及中药复方的影响

目的:研究慢性束缚应激时大鼠脑内糖皮质激素受体的变化以及逍遥散、四君子汤、金匮肾气丸三种中药复方对其影响.方法:制作大鼠束缚应激模型,用特制束缚架连续束缚7 d与21 d,每天3 h用免疫组织化学方法结合图像分析检测中枢(海马CA1区、齿状回、大脑皮质)糖皮质激素受体的变化.结果:慢性束缚应激后,大鼠海马CA1区、大脑皮层和齿状回GR免疫反应阳性细胞平均总面积和阳性细胞数目在慢性应激的早期(7d模型组)明显增多(P＜0.05),免疫反应强度明显增强(P＜0.01).在慢性应激的后期(21 d模型组),则表现为相关脑区GR免疫反应阳性细胞平均总面积和阳性细胞数目均明显减少(P＜0.05),免疫反应强度明显减弱(P＜0.01).中药复方各组相关脑区神经元GR免疫反应阳性细胞平均总面积和阳性细胞数目较21 d模型组明显增高,免疫反应强度明显增强,三给药组之间并无明显差异,说明三给药组均能使GR含量保持于较高的状态,同时能保持GR免疫活性,其中又以逍遥散作用为明显.结论:逍遥散、四君子汤和金匮肾气丸明显逆转糖皮质激素受体下降趋势.     

Altered interaction between PSD-95 and the NMDA receptor following transient global ischemia

The postsynaptic density (PSD) is a cytoskeletal specialization involved in the anchoring of neurotransmitter receptors and in regulating the response of postsynaptic neurons to synaptic stimulation. The postsynaptic protein PSD-95 binds to NMDA receptor subunits NR2A and NR2B and to signaling molecules such as neuronal nitric oxide synthase and p135synGAP. We investigated the effects of transient cerebral ischemia on protein interactions involving PSD-95 and the NMDA receptor in the rat hippocampus. Ischemia followed by reperfusion resulted in a decrease in the solubility of the NMDA receptor and PSD-95 in 1% sodium deoxycholate, the decrease being greater in the vulnerable CA1 hippocampal subfield than in the less sensitive CA3/dentate gyrus regions. Solubilization of the kainic acid receptor GluR6/7 and the PSD-95 binding proteins, neuronal nitric oxide synthase and p135synGAP, also decreased following ischemia. The association between PSD-95 and NR2A and NR2B, as indicated by coimmunoprecipitation, was less in postischemic samples than in sham-operated controls. Ischemia also resulted in a decrease in the size of protein complexes containing PSD-95, but had only a small effect on the size distribution of complexes containing the NMDA receptor. The results indicate that molecular interactions involving PSD-95 and the NMDA receptor are modified by an ischemic challenge.     

Novelty-induced increased expression of immediate-early genes c-fos and arg 3.1 in the mouse brain

The detection of novel stimuli is a memory-dependent process. The presented stimulus has to be compared with memory contents to judge its novelty. In addition, the novelty of stimuli activates attention-related processes that facilitate memory formation. To determine the involvement of limbic and neocortical brain structures in novelty detection, we exposed mice to a novel gustatory stimulus (0.5% saccharin) added to their drinking fluid. We then compared the novelty-induced expression of the two immediate-early genes (IEGs) c-fos and arg 3.1, with their expression in mice familiarized with the same stimulus or mice not exposed to that stimulus. Exposure to taste novelty increased expression of c-fos and arg 3.1 mRNA in the cingulate cortex and deep layers of the parietal cortex. In addition, c-fos mRNA expression was increased in the amygdala and arg 3.1 mRNA was increased in the dentate gyrus. Expression of c-fos and arg 3.1 was elevated 30 min after the exposure to novelty. For arg 3.1, a second peak of expression was found 4.5 h after presentation of the novel stimulus. Our results indicate that the amygdala, the dentate gyrus, and the cingulate and parietal cortices may be involved in novelty detection and associated cognitive events, and suggest that c-fos and arg 3.1 play distinct roles in these processes.     

Hippocampal glucocorticoid receptor expression in the tree shrew: Regulation by psychosocial conflict

Summary 1. This study was conducted to determine whether chronic psychosocial conflict alters the expression of glucocorticoid receptor (GR) mRNA in the hippocampus of male tree shrews (Tupaia belangeri).2. To generate probes for thein situ hybridization, the tree shrew GR gene was partly cloned. There was a 90% homology between the deduced amino acid sequence of the cloned tree shrew GR and that of the corresponding human GR sequence.³⁵S-Labeled riboprobes which had been transcribed from the tree shrew GR clone hybridized to pyramidal neurons in all subregions of the tree shrew hippocampal formation and to granule neurons in the dentate gyrus.3. Afterin situ hybridization, the expression of GR mRNA was semiquantitatively determined by counting silver grains over single neurons of the hippocampal formation of psychosocially stressed tree shrews and control animals. After 12 days of social conflict, the number of silver grains in the CA1 and CA3 pyramidal neurons of stressed animals was significantly lower than in controls. No statistically significant differences in mRNA expression were observed in the pyramidal neurons of the subiculum and in the granule neurons of the dentate gyrus.4. The present results suggest that psychosocial stress leads to a site-specific down-regulation of hippocampal GR via modification of mRNA expression.     

Protein kinase C phosphorylation regulates membrane insertion of GABAA receptor subtypes that mediate tonic inhibition

Tonic inhibition in the brain is mediated largely by specialized populations of extrasynaptic receptors, γ-aminobutyric acid receptors (GABA(A)Rs). In the dentate gyrus region of the hippocampus, tonic inhibition is mediated primarily by GABA(A)R subtypes assembled from α4β2/3 with or without the δ subunit. Although the gating of these receptors is subject to dynamic modulation by agents such as anesthetics, barbiturates, and neurosteroids, the cellular mechanisms neurons use to regulate their accumulation on the neuronal plasma membrane remain to be determined. Using immunoprecipitation coupled with metabolic labeling, we demonstrate that the α4 subunit is phosphorylated at Ser(443) by protein kinase C (PKC) in expression systems and hippocampal slices. In addition, the β3 subunit is phosphorylated on serine residues 408/409 by PKC activity, whereas the δ subunit did not appear to be a PKC substrate. We further demonstrate that the PKC-dependent increase of the cell surface expression of α4 subunit-containing GABA(A)Rs is dependent on Ser(443). Mechanistically, phosphorylation of Ser(443) acts to increase the stability of the α4 subunit within the endoplasmic reticulum, thereby increasing the rate of receptor insertion into the plasma membrane. Finally, we show that phosphorylation of Ser(443) increases the activity of α4 subunit-containing GABA(A)Rs by preventing current run-down. These results suggest that PKC-dependent phosphorylation of the α4 subunit plays a significant role in enhancing the cell surface stability and activity of GABA(A)R subtypes that mediate tonic inhibition.     

Transient ischemia enhances tyrosine phosphorylation and binding of the NMDA receptor to the Src homology 2 domain of phosphatidylinositol 3-kinase in the rat hippocampus

Tyrosine phosphorylation of the NMDA receptor has been implicated in the regulation of the receptor channel. We investigated the effects of transient (15 min) global ischemia on tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B, and the interaction of NR2 subunits with the SH2 domain of phosphatidylinositol 3-kinase (PI3-kinase) in vulnerable CA1 and resistant CA3/dentate gyrus of the hippocampus. Transient ischemia induced a marked increase in the tyrosine phosphorylation of NR2A in both regions. The tyrosine phosphorylation of NR2B in CA3/dentate gyrus after transient ischemia was sustained and greater than that in CA1. PI3-kinase p85 was co-precipitated with NR2B after transient global ischemia. The SH2 domain of the p85 subunit of PI3-kinase bound to NR2B, but not to NR2A. Binding to NR2B was increased following ischemia and the increase in binding in CA3/dentate gyrus (4.5-fold relative to sham) was greater than in CA1 (1.7-fold relative to sham) at 10 min of reperfusion. Prior incubation of proteins with an exogenous protein tyrosine phosphatase or with a phosphorylated peptide (pYAHM) prevented binding. The results suggest that sustained increases in tyrosine phosphorylation and increased interaction of NR2B with the SH2 domain of PI3-kinase may contribute to altered signal transduction in the CA3/dentate gyrus after transient ischemia.     

Effects of protein kinase C modulation on NMDA receptor mediated regulation of neurotransmitter enzyme and c-fos protein in cultured neurons

The role of protein kinase C (PKC) in N-methyl-d-aspartate (NMDA) receptor-mediated biochemical differentiation and c-fos protein expression was investigated in cultured cerebellar granule neurons. The biochemical differentiation of glutamatergic granule cells was studied in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When the partially depolarized cells were treated with NMDA for the last 1 to 3 days (between 2 and 5 days in vitro), it elevated the specific activity of glutaminase. In contrast, NMDA had little effect on the activity of aspartate aminotransferase or of lactate dehydrogenase. Treatment of 10-day old granule neurons with NMDA also resulted in a marked increase in the immunocytochemically measured expression of c-fos protein. The increases in both the activity of glutaminase and the steady state level of c-fos protein were specific to the activation of NMDA receptors, as they were completely blocked byd,l-2-amino-5-phosphonovaleric acid. The specific stimulation of NMDA receptors in PKC-depleted granule neurons or in the presence of reasonably specific PKC inhibitors also produced significant elevation in the activity of glutaminase and the expression of c-fos protein. These increases were similar in magnitude to those observed in the granule neurons of the respective control groups. Our findings demonstrate that PKC is not directly involved in the NMDA receptor-mediated signal transduction processes associated with biochemical differentiation and c-fos induction in cerebellar granule neurons.     

Steroid Hormone Actions on the Brain: When Is the Genome Involved?

It has become customary to distinguish between so-called "genomic" actions of steroid hormones involving intracellular receptors and "non-genomic" effects of steroids that involve putative cell surface receptors. Whereas there is no doubt that this distinction has considerable validity, it does not go far enough in addressing the variety of mechanisms that steroid hormones use to produce their effects on cells. This is because cell surface receptors may signal changes in gene expression, while genomic actions sometimes affect neuronal excitability, often doing so quite rapidly. Moreover, steroid hormones and neurotransmitters may operate together to produce effects, and sometimes these effects involve collaborations between groups of neurons. As illustrations. evidence is reviewed in this article that a number of steroid actions in the hippocampus involves the co-participation of excitatory amino acids. These interactions are evident for the regulation of synaptogenesis by estradiol in the CA1 pyramidal neurons or hippocampus and for the induction of dendritic atrophy of CA3 neurons by repeated stress as well as by glucocorticoid injections. In addition, neurogenesis in the adult and developing dentate gyrus is "contained" by adrenal steroids as well as by excitatory amino acids. In each of these three examples, NMDA receptors are involved. These results not only point to a high degree of interdependency between certain neurotransmitters and the actions of steroid hormones but also emphasize the degree to which structural plasticity is an important aspect of steroid hormone action in the adult as well as developing nervous system.     

非基因型雌激素膜性受体GPR30在生后雌性大鼠海马内的发育学表达与亚细胞水平定位研究

为了研究非基因型雌激素膜性受体GPR30对海马的结构和功能的调节作用,应用硫酸镍铵增强显色的免疫组化技术以及酶标免疫电镜技术,观察了生后雌性大鼠海马内GPR30表达的变化及其免疫阳性产物在神经元亚细胞水平的定位情况．结果显示,GPR30免疫阳性产物主要位于海马CA区的锥体层神经元与齿状回颗粒层的神经元内,其表达水平随发育呈增加趋势．P0时在雌性大鼠海马未发现明显GPR30免疫阳性反应,P7后免疫阳性物质开始在CA2出现,P14时见于 CA1、CA2和齿状回,P30和P60主要见于CA1、CA2、CA3和齿状回．在光镜下,GPR30免疫阳性产物位于细胞核外的胞浆中,细胞核未见免疫阳性反应．在透射电镜下可见其位于神经元的胞浆内,可能主要是粗面内质网,也可见于线粒体和细胞膜．以上结果证实,GPR30是一种位于细胞核外的、非基因型作用的雌激素受体,可能参与了雌激素对海马锥体神经元突触可塑性和学习记忆等功能的调节,还可能参与了对齿状回成年神经干细胞某些活动的调节．