Estrogen synthesis in the brain—Role in synaptic plasticity and memory

Estrogen and androgen are synthesized from cholesterol locally in hippocampal neurons of adult animals. These neurosteroids are synthesized by cytochrome P450s and hydroxysteroid dehydrogenases (HSDs) and 5alpha-reductase. The expression levels of enzymes are as low as 1/200–1/50,000 of those in endocrine organs, however these numbers are high enough for local synthesis. Localization of P450(17alpha), P450arom, 17beta-HSD and 5alpha-reductase is observed in principal glutamatergic neurons in CA1, CA3 and the dendate gyrus. Several nanomolar levels of estrogen and androgen are observed in the hippocampus.Estrogen modulates memory-related synaptic plasticity not only slowly but also rapidly in the hippocampus. Rapid action of 17beta-estradiol via membrane receptors is demonstrated for spinogenesis and long-term depression (LTD). The enhancement of LTD by 1–10 nM estradiol occurs within 1 h. The density of spine is increased in CA1 pyramidal neurons within 2 h after application of estradiol. The density of spine-like structure is, however, decreased by estradiol in CA3 pyramidal neurons. ERalpha, but not ERbeta, induces the same enhancement/suppression effects on both spinogenesis and LTD.     

Local neurosteroid production in the hippocampus: influence on synaptic plasticity of memory

In neuroendocrinology, it is believed that steroid hormones are synthesized in the gonads and/or adrenal glands, and reach the brain via the blood circulation. In contrast to this view, we are in progress of demonstrating that estrogens and androgens are also synthesized locally by cytochrome P450s in the hippocampus, and that these steroids act rapidly to modulate neuronal synaptic plasticity. We demonstrated that estrogens were locally synthesized in the adult hippocampal neurons. In the pathway of steroidogenesis, cholesterol is converted to pregnenolone (by P450scc), dehydroepiandrosterone [by P450(17alpha)], androstenediol (by 17beta-hydroxysteroid dehydrogenase, 17beta-HSD), testosterone (by 3beta-HSD) and finally to estradiol (by P450arom) and dihydrotestosterone (by 5alpha-reductase). The basal concentration of estradiol in the hippocampus was approximately 1 nM, which was greater than that in blood plasma. Significant expression of mRNA for P450scc, P450(17alpha), P450arom, 17beta-HSD, 3beta-HSD and 5alpha-reductase was demonstrated by RT-PCR. Their mRNA levels in the hippocampus were 1/200-1/5,000 of those in the endocrine organs. Localization of P450(17alpha) and P450arom was observed in synapses in addition to endoplasmic reticulum of principal neurons using immunoelectron microscopy. Different from slow action of gonadal estradiol which reaches the brain via the blood circulation, hippocampal neuron-derived estradiol may act locally and rapidly within the neurons. For example, 1 nM 17beta-estradiol rapidly enhanced the long-term depression (LTD) not only in CA1 but also in CA3 and dentate gyrus. The density of thin spines was selectively increased within 2 h upon application of 1 nM estradiol in CA1 pyramidal neurons. Only ERalpha agonist propyl-pyrazole-trinyl-phenol induced the same enhancing effect as estradiol on both LTD and spinogenesis in the CA1. ERbeta agonist hydroxyphenyl-propionitrile suppressed LTD and did not affect spinogenesis. Localization of estrogen receptor ERalpha in spines in addition to nuclei of principal neurons implies that synaptic ERalpha can drive rapid modulation of synaptic plasticity by endogenous estradiol.     

Adult male rat hippocampus synthesizes estradiol from pregnenolone by cytochromes P45017alpha and P450 aromatase localized in neurons

In adult mammalian brain, occurrence of the synthesis of estradiol from endogenous cholesterol has been doubted because of the inability to detect dehydroepiandrosterone synthase, P45017alpha. In adult male rat hippocampal formation, significant localization was demonstrated for both cytochromes P45017alpha and P450 aromatase, in pyramidal neurons in the CA1-CA3 regions, as well as in the granule cells in the dentate gyrus, by means of immunohistochemical staining of slices. Only a weak immunoreaction of these P450s was observed in astrocytes and oligodendrocytes. ImmunoGold electron microscopy revealed that P45017alpha and P450 aromatase were localized in pre- and postsynaptic compartments as well as in the endoplasmic reticulum in principal neurons. The expression of these cytochromes was further verified by using Western blot analysis and RT-PCR. Stimulation of hippocampal neurons with N-methyl-d-aspartate induced a significant net production of estradiol. Analysis of radioactive metabolites demonstrated the conversion from [(3)H]pregnenolone to [(3)H]estradiol through dehydroepiandrosterone and testosterone. This activity was abolished by the application of specific inhibitors of cytochrome P450s. Interestingly, estradiol was not significantly converted to other steroid metabolites. Taken together with our previous finding of a P450scc-containing neuronal system for pregnenolone synthesis, these results imply that 17beta-estradiol is synthesized by P45017alpha and P450 aromatase localized in hippocampal neurons from endogenous cholesterol. This synthesis may be regulated by a glutamate-mediated synaptic communication that evokes Ca(2+) signals.     

Rapid increases in immature synapses parallel estrogen-induced hippocampal learning enhancements

Dramatic increases in hippocampal spine synapse density are known to occur within minutes of estrogen exposure. Until now, it has been assumed that enhanced spinogenesis increased excitatory input received by the CA1 pyramidal neurons, but how this facilitated learning and memory was unclear. Delivery of 17β-estradiol or an estrogen receptor (ER)-α (but not ER-β) agonist into the dorsal hippocampus rapidly improved general discrimination learning in female mice. The same treatments increased CA1 dendritic spines in hippocampal sections over a time course consistent with the learning acquisition phase. Surprisingly, estrogen-activated spinogenesis was associated with a decrease in CA1 hippocampal excitatory input, rapidly and transiently reducing CA1 AMPA activity via a mechanism likely reflecting AMPA receptor internalization and creation of silent or immature synapses. We propose that estrogens promote hippocampally mediated learning via a mechanism resembling some of the broad features of normal development, an initial overproduction of functionally immature connections being subsequently “pruned” by experience.Estradiol rapidly and dramatically increases hippocampal dendritic spine and synapse density within minutes of application (1–4). There is a strong correlative association between estrogen-induced spinogenesis and improvements in cognition (5); however, the relationship of these structural changes to estrogen-induced alterations in hippocampal function is unclear. Our laboratory recently reported that the density of hippocampal CA1 pyramidal dendritic spines increases very rapidly after systemic treatment with 17β-estradiol or estrogen receptor (ER) -selective agonists in ovariectomized female mice, changes that are paralleled by learning enhancements (2, 3). Estrogen-induced rapid structural changes are substantial, increasing spine density by 30–50% within 15–40 min of hormone application (1–3, 6). As a result, adult rodents can experience the addition of thousands of CA1 synapses within a span of minutes after exposure to estradiol. These effects of estrogens reproduce the changes occurring during the 4-d estrous cycle of female rodents, which include the induction of CA1 spines (7).How these processes contribute to the behavioral changes observed after estradiol treatment is not understood. Estradiol enhances excitatory neurotransmission throughout the hippocampus (8–10), and activates BDNF signaling in the mossy fiber system (11). Dendritic spines turn over more rapidly in the hippocampus than in the neocortex (12), particularly in the case of estradiol-induced spines (13). Such rapid, transient, and apparently indiscriminate increases in excitatory synapse formation would seem, at first sight, to be more likely to interfere with preexisting brain circuits and impair normal information processing than to enhance cognitive function.How then, does enhancement of spine formation lead to improved cognitive function? To address this question, we focused specifically on the effects of estradiol in the dorsal CA1 hippocampus, as a site mediating the rapid improving effects of estrogens on learning. Estradiol application, via a mechanism involving ERα, rapidly increases dendritic spine density in the CA1 pyramidal stratum oriens and stratum radiatum subregions. However, contrary to our initial assumptions based on previous work in this field, increased spinogenesis did not result in increased CA1 excitatory input. Rather, estrogen-induced formation of hippocampal spines was associated with a decrease in the ability of CA1 neurons to respond to AMPA receptor (AMPAR) activation, resulting in decreased excitatory input to CA1 neurons. This appears to be the result of AMPAR internalization from the synaptic membrane (6). Taken together, our data suggest that estrogens induce formation of “silent” or immature synapses that act as a substrate for the storage of new memories (14, 15). This finding explains estrogens’ ability to rapidly improve learning without precipitating uncontrolled activation of the hippocampal circuitry. These effects of estradiol on CA1 pyramidal neurons phenotypically resemble the structural and functional properties of neurons during development, when neurons with higher levels of spine density and higher numbers of silent/immature synapses are present before the activity dependent refinement of neural circuitry.     

Sex differences in hippocampal estradiol-induced N-methyl-D-aspartic acid binding and ultrastructural localization of estrogen receptor-alpha

Estradiol increases dendritic spine density and synaptogenesis in the CA1 region of the female hippocampus. This effect is specific to females, as estradiol-treated males fail to show increases in hippocampal spine density. Estradiol-induced spinogenesis in the female is dependent upon upregulation of the N-methyl-D-aspartic acid (NMDA) receptor as well as on non-nuclear estrogen receptors (ER), including those found in dendrites. Thus, in the male, the inability of estradiol to induce spinogenesis may be related to a failure of estradiol to increase hippocampal NMDA receptors as well as a paucity of dendritic ER. In the first experiment, we sought to investigate this possibility by assessing NMDA receptor binding, using [(3)H]-glutamate autoradiography, in estradiol-treated males and females. We found that while estradiol increases NMDA binding in gonadectomized females, estradiol fails to modulate NMDA binding in gonadectomized males. To further investigate sex differences in the hippocampus, we conducted a second separate, but related, ultrastructural study in which we quantified ERalpha-immunoreactivity (ERalpha-ir) in neuronal profiles in the CA1 region of the hippocampus in intact males and females in diestrus and proestrus. Consistent with previous reports in the female, we found ERalpha-ir in several extranuclear sites including dendrites, spines, terminals and axons. Statistical analyses revealed that females in proestrus had a 114.3% increase in ERalpha-labeled dendritic spines compared to females in diestrus and intact males. Taken together, these studies suggest that both the ability of estrogen to increase NMDA binding in the hippocampus and the presence of ERalpha in dendritic spines may contribute to the observed sex difference in estradiol-induced hippocampal spinogenesis.     

Down-regulation of neurosteroid biosynthesis in corticolimbic circuits mediates social isolation-induced behavior in mice

Allopregnanolone (ALLO), synthesized by pyramidal neurons, is a potent positive allosteric modulator of the action of GABA at GABA(A) receptors expressing specific neurosteroid binding sites. In the brain, ALLO is synthesized from progesterone by the sequential action of two enzymes: 5alpha-reductase type I (5alpha-RI) and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD). In the cortex, hippocampus, and amygdala, these enzymes are colocalized in principal glutamatergic output neurons [Agís-Balboa RC, Pinna G, Zhubi A, Maloku E, Veldic M, Costa E, Guidotti A (2006) Proc Natl Acad Sci USA 103:14602-14607], but they are not detectable in GABAergic interneurons. Using RT-PCR and in situ hybridization, this study compares 5alpha-RI and 3alpha-HSD mRNA brain expression levels in group housed and in socially isolated male mice for 4 weeks. In these socially isolated mice, the mRNA expression of 5alpha-RI was dramatically decreased in hippocampal CA3 glutamatergic pyramidal neurons, dentate gyrus granule cells, glutamatergic neurons of the basolateral amygdala, and glutamatergic pyramidal neurons of layer V/VI frontal (prelimbic, infralimbic) cortex (FC). In contrast, 5alpha-RI mRNA expression failed to change in CA1 pyramidal neurons, central amygdala neurons, pyramidal neurons of layer II/III FC, ventromedial thalamic nucleus neurons, and striatal medium spiny and reticular thalamic nucleus neurons. Importantly, 3alpha-HSD mRNA expression was unchanged by protracted social isolation (Si). These data suggest that, in male mice, after 4 weeks of Si, the expression of 5alpha-RI mRNA, which is the rate-limiting-step enzyme of ALLO biosynthesis, is specifically down-regulated in glutamatergic pyramidal neurons that converge on the amygdala from cortical and hippocampal regions. In socially isolated mice, this down-regulation may account for the appearance of behavioral disorders such as anxiety, aggression, and cognitive dysfunction.     

Nanomolar dose of bisphenol A rapidly modulates spinogenesis in adult hippocampal neurons

We demonstrated the rapid effects of 10nM bisphenol A (BPA) on the spinogenesis of adult rat hippocampal slices. The density of spines was analyzed by imaging Lucifer Yellow-injected CA1 neurons in slices. Not only the total spine density but also the head diameter distribution of spine was quantitatively analyzed. Spinogenesis was significantly enhanced by BPA within 2h. In particular, the density of middle-head spine (with head diameter of 0.4-0.5μm) was significantly increased. Hydroxytamoxifen, an antagonist of both estrogen-related receptor gamma (ERRγ) and estrogen receptors (ERα/ERβ), blocked the BPA-induced enhancement of the spine density. However, ICI 182,780, an antagonist of ERα/ERβ, did not suppress the BPA effects. Therefore, ERRγ is deduced to be a high affinity receptor of BPA, responsible for modulation of spinogenesis. The BPA-induced enhancement of spinogenesis was also suppressed by MAP kinase inhibitor, PD98059, and the blocker of NMDA receptors, MK-801. Washout of BPA for additional 2h after 2h BPA treatment abolished the BPA-induced enhancement of spinogenesis, suggesting that the BPA effect was reversible. ERRγ was localized at synapses as well as cell bodies of principal neurons. ERRγ at synapses may contribute to the observed rapid effect. The level of BPA in the hippocampal slices was determined by mass-spectrometric analysis.     

Estrogen and ovariectomy regulate mRNA and protein of glutamic acid decarboxylases and cation-chloride cotransporters in the adult rat hippocampus

17beta-Estradiol spatiotemporally regulates the gamma-aminobutyric acid (GABAergic) tone in the adult hippocampus. However, the complex estrogenic effect on the GABAergic system is still unclear. In adult central nervous system (CNS) neurons, GABA can induce both inhibitory and excitatory actions, which are predominantly controlled by the cation-chloride cotransporters NKCC1 and KCC2. We therefore studied the estrogenic regulation of two glutamate decarboxylase (GAD) isoforms, GAD65 and GAD67, as well as NKCC1 and KCC2 in the adult female rat hippocampus by immunohistochemistry and in situ hybridization. First, we focused on the duration after ovariectomy (OVX) and its effects on GAD65 protein levels. The basal number of GAD65-immunoreactive cells decreased after long-term (10 days) OVX compared to short-term (3 days) OVX. We found that, only after long-term OVX but not after short-term OVX, estradiol increased the number of GAD65-immunoreactive cells in the CA1 pyramidal cell layer. Furthermore, estradiol did not alter the GAD65-immunoreactive cell population in any other CA1 subregion. Second, we therefore focused on long-term OVX and the estrogenic regulation of GAD and cation-chloride cotransporter mRNA levels. In the pyramidal cell layer, estradiol affected GAD65, GAD67 and NKCC1 mRNA levels, but not KCC2 mRNA levels. Both GAD65 and NKCC1 mRNA levels increased within 24 h after estradiol treatment, followed by a subsequent increase in GAD67 mRNA levels. These findings suggest that basal levels of estrogen might contribute to a balance between the excitatory and inhibitory synaptic transmission onto CA1 pyramidal cells by regulating perisomatic GAD and NKCC1 expression in the adult hippocampus.     

Ⅱ组代谢型谷氨酸受体阻断剂对大鼠海马神经元凋亡的影响

目的观察Ⅱ组代谢型谷氨酸受体阻断剂对大鼠海马神经元凋亡的影响。方法 SD大鼠24只随机分为假手术组、痴呆组和阻断剂组,每组8只。大鼠脑室注射凝聚肽Aβ_(25-35)5建立痴呆大鼠模型,阻断剂组1周后行阻断剂脑室注射,另2组等量注射人工脑脊液4周行Morris水迷宫测试;测试1周后取材行病理观察,采用TUNEL法检测海马CA1区锥体细胞凋亡情况。结果与假手术组比较,痴呆组大鼠平均潜伏期明显延长,平台象限滞留时间明显缩短(P0.05);与痴呆组比较,阻断剂组大鼠上述指标明显提高(P0.05)。与假手术组比较,痴呆组大鼠海马CA1区可见大量的凋亡锥体细胞,阳性锥体细胞数、总面积、平均吸光度(A)值明显升高(P0.05);与痴呆组比较,阻断剂组大鼠海马CA1区可见明显的阳性细胞和核固缩,但其阳性染色锥体细胞数、总面积、平均A值明显降低(P0.05)。结论Ⅱ组代谢型谷氨酸受体阻断剂可明显抑制痴呆引起的海马CA1区锥体细胞的凋亡,提示其可能通过影响细胞凋亡,参与痴呆的发病过程。     

Age-related anatomical changes in the rat hippocampus: retardation by choline alfoscerate treatment

The age-related anatomical changes in the rat hippocampus were evaluated in male Sprague-Dawley rats of 3 (young), 12 (mature) and 24 (aged) months by counting the number of nerve cells in the CA1 and CA3 fields and in the dentate gyrus and by measuring the density of Nissl bodies in the cytoplasm of the pyramidal and granule neurons of the above areas. Moreover, the effect of 3 months choline alfoscerate treatment on the anatomical parameters examined was evaluated. The number of pyramidal neurons of the CA1 field and of granule neurons of the dentate gyrus was not significantly changed between young and mature animals, but it was decreased in aged rats. The number of pyramidal neurons of the CA3 field showed a progressive age-dependent reduction. The density of Nissl bodies was the highest in the cytoplasm of pyramidal or granule neurons in mature rats followed in descending order by young and aged animals. Choline alfoscerate treatment counteracted the age-related loss of nerve cells in the 3 hippocampal portions examined and slow-drown the decrease of Nissl bodies in the cytoplasm of pyramidal or of granule neurons in the hippocampus. The significance of changes induced by choline alfoscerate in the hippocampus of aged rats and the possible mechanism of action of the compound are discussed.     

Feedforward excitation of the hippocampus by afferents from the entorhinal cortex: redefinition of the role of the trisynaptic pathway.

For the past 3 decades, functional characterizations of the hippocampus have emphasized its intrinsic trisynaptic circuitry, which consists of successive excitatory projections from the entorhinal cortex to the dentate gyrus, from granule cells of the dentate to the CA3/4 pyramidal cell region, and from CA3/4 to the CA1/2 pyramidal cell region. Despite unequivocal anatomical evidence for a monosynaptic projection from entorhinal to CA3 and CA1/2, few in vivo electrophysiological studies of the direct pathway have been reported. In the experiments presented here, we stimulated axons of entorhinal cortical neurons in vivo and recorded evoked single unit and population spike responses in the dentate, CA3, and CA1 of hippocampus, to determine if pyramidal cells are driven primarily via the monosynaptic or trisynaptic pathways. Our results show that neurons within the three subfields of the hippocampus discharge simultaneously in response to input from a given subpopulation of entorhinal cortical neurons and that the initial monosynaptic excitation of pyramidal cells then is followed by weaker excitatory volleys transmitted through the trisynaptic pathway. In addition, we found that responses of CA3 pyramidal cells often precede those of dentate granule cells and that excitation of CA3 and CA1 pyramidal cells can occur in the absence of granule cell excitation. In total, these results argue for a different conceptualization of the functional organization of the hippocampus with respect to the propagation of activity through its intrinsic pathways: input from the entorhinal cortex initiates a two-phase feedforward excitation of pyramidal cells, with the dentate gyrus providing feedforward excitation of CA3, and with both the dentate and CA3 providing feedforward excitation of CA1.     

Regulation of excitability and plasticity by endocannabinoids and PKA in developing hippocampus

The activity-dependent strengthening and weakening of synaptic transmission are hypothesized to be the basis of not only memory and learning but also the refinement of neural circuits during development. Here we report that, in the developing CA1 area of the hippocampus, endocannabinoid (eCB)-mediated heterosynaptic long-term depression (LTD) of glutamatergic excitatory synaptic transmission is associated with PKA-mediated homosynaptic long-term potentiation (LTP). This form of LTD was dominant at postnatal days 2-10 (P2-P10), attenuated during development, and finally disappeared in the mature hippocampus. Heterosynaptic LTD of excitatory postsynaptic currents in the developing hippocampus was expressed presynaptically, spread to neighboring neurons, and was mediated by eCBs. Heterosynaptic LTD of field excitatory postsynaptic potentials was associated with a decrease in fiber volley amplitude with a similar time course. Depression of fiber volleys was blocked by K(+) channel blockers, suggesting the involvement of the decrease in presynaptic excitability in heterosynaptic LTD. In the P2-P5 hippocampus, eCBs also attenuate LTP and fiber volleys in homosynaptic pathways and help to prevent too much excitability in the neonatal hippocampus where the GABAergic system is poorly developed and even excitatory. In the hippocampus older than P6 (P > 6), however, LTP is protected from eCB-mediated depression by PKA activated at presynaptic sites by high-frequency stimulation, serving to highlight PKA-mediated LTP by weakening inactive synapses even in adjacent cells. Thus, eCBs and PKA make synapses plastic without changing excitability homeostasis in the developing hippocampus.     

Glutamic acid decarboxylase messenger ribonucleic acid is regulated by estradiol and progesterone in the hippocampus.

Ovarian steroids modulate learning, memory, and epileptic seizure activity, functions that are mediated in part by the hippocampus. Normal function depends on precise interactions between the inhibitory gamma-aminobutyric acid (GABA)ergic and excitatory glutamatergic neurons of the hippocampus. To determine whether estradiol and progesterone interact with GABAergic neurons, the levels of mRNA for glutamic acid decarboxylase (GAD), the rate-limiting enzyme for GABA synthesis, were measured by in situ hybridization histochemistry with 35S-labeled riboprobes complimentary to the feline GAD cDNA. The levels of mRNA for GAD were analyzed in selected region of the dorsal hippocampus and medial basal hypothalamus in ovariectomized, ovariectomized estradiol-treated, and ovariectomized estradiol- and progesterone-treated rats. In estradiol-treated rats, GAD mRNA levels increased in GABAergic neurons associated with the CA1 pyramidal cell layer, but not in the stratum oriens of CA1 or any other region of the hippocampus. Estradiol plus progesterone treatment reversed the estradiol-induced increase in GAD mRNA in CA1 and induced a small decrease in the hilus. No effect of estradiol or progesterone was observed in the dorsomedial, ventromedial, or arcuate nuclei of the hypothalamus. Estradiol or progesterone may alter cognitive performance and seizure activity by increasing or decreasing, respectively, the activity of GABAergic neurons in the hippocampus.     

The 17alpha and 17beta isomers of estradiol both induce rapid spine synapse formation in the CA1 hippocampal subfield of ovariectomized female rats

Previous studies have demonstrated that estradiol-17beta and estradiol-17alpha both induce short-latency effects on spatial memory in rats, estradiol-17alpha being at least as potent as its 17beta isomer. To determine whether the mechanisms underlying these behavioral responses might include effects on hippocampal synaptic plasticity, CA1 pyramidal spine synapse density (PSSD) was measured in ovariectomized rats within the first few hours after s.c. estrogen injection. PSSD increased markedly (by 24%) 4.5 h after the administration of 45 microg/kg estradiol-17beta. The PSSD response was significantly greater (44% above control) 30 min after estradiol-17beta injection and was markedly dose dependent; a 3-fold lower estradiol-17beta dose (15 microg/kg) did not significantly affect CA1 PSSD at either 30 min or 4.5 h. Estradiol-17alpha was a more potent inducer of PSSD than estradiol-17beta. Dose-response analysis determined an ED50 for the effect of estradiol-17alpha on PSSD of 8.92 +/- 1.99 microg/kg, with a maximal response at 15 microg/kg. These results demonstrate that high doses of estradiol induce rapid changes in CA1 PSSD. CA1 spine synapse formation appears to be more sensitive to estradiol-17alpha than to estradiol-17beta, paralleling previous data on the effects of these two steroids on spatial memory. Rapid remodeling of hippocampal synaptic connections may thus contribute to the enhancement of spatial mnemonic processing observed within the first few hours after estrogen treatment. The potency of estradiol-17alpha suggests that hormone replacement therapy using this steroid might be useful clinically in ameliorating the impact of low endogenous estrogen production on the development and progression of neurodegenerative disorders involving the hippocampus.     

大鼠海马神经元及其线粒体增龄性改变的形态计量分析

目的探讨大鼠海马神经元及其线粒体形态结构增龄性改变的程度及性质。方法应用组织化学及电镜技术，通过计算机图像分析系统对海马神经元及其线粒体结构进行形态计量分析。结果海马ＣＡ１和ＣＡ３区神经元随增龄出现细胞皱缩，ＣＡ３区神经元数密度在老年组较青年组显著减少（Ｐ＜０．０５），ＣＡ１区神经元数密度各月龄组之间差异无显著性（Ｐ＞０．０５）。ＣＡ３区神经元胞体内线粒体体密度、数密度、比表面及嵴膜密度随增龄而减少，线粒体平均体积及平均截面积随增龄而增大。结论海马神经元及其线粒体形态结构随增龄发生显著性改变，这些形态结构的改变可能是大鼠海马老化的指征。     

Generation of Neurons in the Rat Dentate Gyrus and Hippocampus: Effects of Prenatal and Postnatal Treatment with Ethanol

Neurons in the rat hippocampal formation (the dentate gyrus and the hippocampus) are born over a protracted period, from gestational day (G) 15 into adulthood. Dentate gyral neurons born prenatally are generated from the ventricular zone, whereas those born postnatally are derived from a secondary proliferative zone, the intrahilar zone. In contrast, hippocampal pyramidal neurons are generated only prenatally from the ventricular zone. In the neocortex, ethanol depresses the proliferation of cells in the ventricular zone and stimulates the proliferation of cells in the secondary proliferative zone. The present study tests the hypotheses that prenatal treatment with ethanol has a different effect on the generation of dentate gyral neurons than does postnatal ethanol treatment, and that these differences are determined by the timing of the ethanol exposure relative to the period and site of neuronal generation. Rats were treated with ethanol between G6 and G21 or between postnatal day (P) 4 and P12. They were given an injection of [³H]thymidine on G15, G18, G21, P6, P9, or P12. Rats were killed on P30–P35. The tissue was processed by standard autoradiographic methods and assessed using rigorous stereological procedures. The total number of neurons and the density of radiolabeled neurons in both the dentate gyrus and the CA1 region of the hippocampus were determined. Prenatal ethanol treatment decreased the total number of neurons in the CA1 segment of the hippocampus and had little impact on neuronal number in the dentate gyrus. Likewise, the number of hippocampal and dentate gyral neurons generated daily was significantly lower in ethanol-treated rats than in controls. Postnatal treatment to ethanol, however, significantly increased the total number of dentate gyral neurons and the density of neurons generated postnatally. These postnatal changes depended on the blood ethanol concentration (BEC). At moderate BECs, the total number of neurons in the dentate gyrus and the number of neurons generated was increased. At high BECs, however, neuronal number and neuronal generation were decreased. Postnatal ethanol treatment had no effect on the number of (total or radiolabeled) CA1 neurons. Thus, pre- and postnatal exposure to ethanol have opposite effects both on the number of neurons in the dentate gyrus and on the generation of neurons. These paradoxical effects likely result from three causes: the differential effects of ethanol on the two proliferative zones, the critical period of neuronal development, and the potentially opposite effects of moderate and high BEC.     

Estrogen increases synaptic connectivity between single presynaptic inputs and multiple postsynaptic CA1 pyramidal cells: a serial electron-microscopic study

Dendritic spines are sites of the vast majority of excitatory synaptic input to hippocampal CA1 pyramidal cells. Estrogen has been shown to increase the density of dendritic spines on CA1 pyramidal cell dendrites in adult female rats. In parallel with increased spine density, estrogen has been shown also to increase the number of spine synapses formed with multiple synapse boutons (MSBs). These findings suggest that estrogen-induced dendritic spines form synaptic contacts with preexisting presynaptic boutons, transforming some previously single synapse boutons (SSBs) into MSBs. The goal of the current study was to determine whether estrogen-induced MSBs form multiple synapses with the same or different postsynaptic cells. To quantify same-cell vs. different-cell MSBs, we filled individual CA1 pyramidal cells with biocytin and serially reconstructed dendrites and dendritic spines of the labeled cells, as well as presynaptic boutons in synaptic contact with labeled and unlabeled (i.e., different-cell) spines. We found that the overwhelming majority of MSBs in estrogen-treated animals form synapses with more than one postsynaptic cell. Thus, in addition to increasing the density of excitatory synaptic input to individual CA1 pyramidal cells, estrogen also increases the divergence of input from individual presynaptic boutons to multiple postsynaptic CA1 pyramidal cells. These findings suggest the formation of new synaptic connections between previously unconnected hippocampal neurons.     

RGS14 is a natural suppressor of both synaptic plasticity in CA2 neurons and hippocampal-based learning and memory

Learning and memory have been closely linked to strengthening of synaptic connections between neurons (i.e., synaptic plasticity) within the dentate gyrus (DG)–CA3–CA1 trisynaptic circuit of the hippocampus. Conspicuously absent from this circuit is area CA2, an intervening hippocampal region that is poorly understood. Schaffer collateral synapses on CA2 neurons are distinct from those on other hippocampal neurons in that they exhibit a perplexing lack of synaptic long-term potentiation (LTP). Here we demonstrate that the signaling protein RGS14 is highly enriched in CA2 pyramidal neurons and plays a role in suppression of both synaptic plasticity at these synapses and hippocampal-based learning and memory. RGS14 is a scaffolding protein that integrates G protein and H-Ras/ERK/MAP kinase signaling pathways, thereby making it well positioned to suppress plasticity in CA2 neurons. Supporting this idea, deletion of exons 2–7 of the RGS14 gene yields mice that lack RGS14 (RGS14-KO) and now express robust LTP at glutamatergic synapses in CA2 neurons with no impact on synaptic plasticity in CA1 neurons. Treatment of RGS14-deficient CA2 neurons with a specific MEK inhibitor blocked this LTP, suggesting a role for ERK/MAP kinase signaling pathways in this process. When tested behaviorally, RGS14-KO mice exhibited marked enhancement in spatial learning and in object recognition memory compared with their wild-type littermates, but showed no differences in their performance on tests of nonhippocampal-dependent behaviors. These results demonstrate that RGS14 is a key regulator of signaling pathways linking synaptic plasticity in CA2 pyramidal neurons to hippocampal-based learning and memory but distinct from the canonical DG–CA3–CA1 circuit.