Localization of copper-zinc superoxide dismutase mRNA in human hippocampus by in situ hybridization

The cellular localization of copper-zinc superoxide dismutase (CuZn SOD) mRNA was determined in the human hippocampus by in situ hybridization with a 35S-labelled DNA probe complementary to human CuZn SOD mRNA. A positive hybridization signal was detected in pyramidal cell layers CA1-CA4 of Ammon's horn (CA), pyramidal cells of subiculum and in the granule cells of the dentate gyrus. The fact that CuZn SOD gene expression is important in neurones which are preferentially vulnerable in neurodegenerative processes such as Alzheimer's disease, suggests a role played by oxygen free radicals in the mechanism of nerve cell death.     

Differences of neuronal sensitivity to amino acids and related compounds in the rat hippocampal slice

The perfusion of slices of rat hippocampus with solutions containing N-methyl-DL-aspartic acid (NMA), kainic acid, ibotenic acid or quinolinic acid produced a reduction in the size of antidromically evoked population spikes in the CA1 pyramidal cells or dentate gyrus granule cells. The relative potencies of these compounds on CA1 cells compared with granule cells were kainate 3.65, quinolinate 3.46, NMA 2.19 and ibotenate 1.50. Since the former two compounds are known to show a degree of selective toxicity towards the CA1 cells, whereas NMA and ibotenate do not, these results are consistent with the excitotoxic hypothesis that excitation and neurotoxicity are related.     

Evidence of functional vitamin D receptors in rat hippocampus

The steroid hormone vitamin D has important biological roles in calcium transport, cell growth, and cell differentiation. Its cellular activities are mediated by high affinity interaction with the vitamin D receptor. In brain, autoradiographic, immunohistologic, and messenger RNA expression studies implicate a number of neuronal systems, including the hippocampus, as potential targets of vitamin D. However, cellular distribution and protein expression, and binding of the receptor to vitamin D response elements have yet to be established in hippocampus. This investigation was undertaken to characterize the vitamin D receptor in rat hippocampus with western blot, immunocytochemistry, and gel shift analyses. The presence of the receptor protein in hippocampus extracts was revealed with western blotting using an anti-rat vitamin D receptor antiserum. In vivo and in vitro immunocytochemical results confirmed the presence of vitamin D receptor in neuronal and glial cells. In the hippocampus, the receptor was localized in pyramidal and granule cell layers, CA1, CA2, and CA3 subfields and in the dentate gyrus. Double labeling for the vitamin D receptor and glial fibrillary acidic protein revealed that glia also expressed the receptor protein. Gel shift analyses evaluated with the murine osteopontin vitamin D response element indicated a specific, bound receptor-containing complex from hippocampal extracts. Altogether, these findings clearly document the localization of vitamin D receptor in rat hippocampus and that hippocampus contains vitamin D receptors capable of specifically binding to DNA. In combination with reports of a neuroprotective role for vitamin D in hippocampal cell survival, these data suggest that the endogenous vitamin D receptor may mitigate processes related to cellular homeostasis, perhaps through a calcium buffering mechanism.     

Recurrent seizures and hippocampal sclerosis following intrahippocampal kainate injection in adult mice: electroencephalography, histopathology and synaptic reorganization similar to mesial temporal lobe epilepsy

Human mesial temporal lobe epilepsy is characterized by hippocampal seizures associated with pyramidal cell loss in the hippocampus and dispersion of dentate gyrus granule cells. A similar histological pattern was recently described in a model of extensive neuroplasticity in adult mice after injection of kainate into the dorsal hippocampus [Suzuki et al. (1995) Neuroscience 64, 665-674]. The aim of the present study was to determine whether (i) recurrent seizures develop in mice after intrahippocampal injection of kainate, and (ii) the electroencephalographic, histopathological and behavioural changes in such mice are similar to those in human mesial temporal lobe epilepsy. Adult mice receiving a unilateral injection of kainate (0.2 microg; 50 nl) or saline into the dorsal hippocampus displayed recurrent paroxysmal discharges on the electroencephalographic recordings associated with immobility, staring and, occasionally, clonic components. These seizures started immediately after kainate injection and recurrid for up to eight months. Epileptiform activities occurred most often during sleep but occasionally while awake. The pattern of seizures did not change over time nor did they secondarily generalize. Glucose metabolic changes assessed by [14C]2-deoxyglucose autoradiography were restricted to the ipsilateral hippocampus for 30 days, but had spread to the thalamus by 120 days after kainate. Ipsilateral cell loss was prominent in hippocampal pyramidal cells and hilar neurons. An unusual pattern of progressive enlargement of the dentate gyrus was observed with a marked radial dispersion of the granule cells associated with reactive astrocytes. Mossy fibre sprouting occurred both in the supragranular molecular layer and infrapyramidal stratum oriens layer of CA3. The expression of the embryonic form of the neural cell adhesion molecule coincided over time with granule cell dispersion. Our data describe the first histological, electrophysiological and behavioural evidence suggesting that discrete excitotoxic lesions of the hippocampus in mice can be used as an isomorphic model of mesial temporal lobe epilepsy.     

Short-term glucocorticoid manipulations affect neuronal morphology and survival in the adult dentate gyrus

In order to determine whether short-term glucocorticoid manipulations influence the morphology and survival of neurons in the adult mammalian hippocampal formation, we performed quantitative analyses of Golgi-impregnated and Nissl-stained tissue from the brains of sham operated male rats, adrenalectomized male rats and adrenalectomized male rats which received corticosterone replacement. Three days after adrenalectomy, massive cell death, as detected by a dramatic increase in number of pyknotic cells, was observed in the granule cell layer of the dentate gyrus. By seven days following adrenalectomy, the numbers of pyknotic cells were even greater. Moreover, significant decreases in cross-sectional cell body area and numbers of dendritic branch points of Golgi-impregnated dentate gyrus granule cells were detected at seven days after adrenalectomy. Replacement of corticosterone to adrenalectomized rats prevented the appearance of large numbers of pyknotic cells as well as the decrease in granule cell cross-sectional cell body area and the numbers of dendritic branch points. In contrast, no obvious signs of degeneration were detected in the pyramidal cell layers of the CA1 and CA3 regions of the hippocampus at either three or seven days following adrenalectomy. In addition, no significant changes in morphological characteristics were observed in CA1 or CA3 pyramidal cells with adrenalectomy. These results show that dentate gyrus granule cells require glucocorticoids for their survival and for the maintenance of normal morphology and suggest that granule cell morphology and/or survival may undergo constant fluctuation in response to diurnal rhythms or stress-induced changes in glucocorticoid levels.     

Histochemical localization of cytochrome oxidase in the hippocampus: correlation with specific neuronal types and afferent pathways

Cytochrome oxidase was histochemically localized in the hippocampus and dentate gyrus of various species of mammals. The most intense staining was observed within stratum moleculare of areas CA1-3 and the outer molecular layer of the dentate gyrus, as well as the somatic and basal dendritic layers of CA3. These regions correspond to the synaptic terminal fields of major excitatory afferent pathways to the hippocampus. The somata of CA3 pyramidal cells and various interneurons were more intensely stained than CA1 pyramidal cells and dentate granule cells, and these levels appeared to correlate positively with their reported rates of spontaneous firing. At the electron-microscopic level, the highest concentrations of densely reactive mitochondria were localized within the distal apical dendritic profiles of principal cells (granule and pyramidal) and certain interneurons (pyramidal basket and stratum pyramidale interneurons). The specific layers in which these structures were found are known to receive intense excitatory input from the perforant pathway. High concentrations of reactive mitochondria were also observed within the somata and proximal dendrites of CA3 pyramidal cells and various interneurons, confirming our light-microscopic observations. These results demonstrated that not only can soma and dendrites of the same cell have disparate but distinct levels of cytochrome oxidase activity, but the pattern of reactivity within a neuron's apical and basal dendrites, or even within specific dendritic segments of the same dendrite can be quite different. While the levels of somatic reactivity correlate with reported levels of spontaneous and/or synaptic activity, the degree of dendritic and somatic staining appeared to be more closely related to the intensity of convergent and/or pathway-specific excitatory synaptic input.     

Dexamethasone induces limited apoptosis and extensive sublethal damage to specific subregions of the striatum and hippocampus: implications for mood disorders

It has been shown previously that the synthetic corticosteroid dexamethasone induces apoptosis of granule cells in the dentate gyrus and striatopallidal neurons in the dorsomedial caudate-putamen. We investigated whether or not dexamethasone can induce damage to other neuronal populations. This issue was addressed using OX42 immunohistochemistry to visualise activated microglia and thereby gauge the extent of dexamethasone-induced neuronal death. A single dose of dexamethasone (20mg/kg, i.p.) administered to young male Sprague-Dawley rats induced a strong microglial reaction which was restricted to the striatum, the dentate gyrus and all of the CA subfields of the hippocampus. Some OX42-immunoreactive cells were also seen in the lateral septal nucleus. Subsequent quantitative analysis of silver/methenamine-stained sections confirmed that acute administration of dexamethasone induced apoptosis in the striatum and all regions of the hippocampus at doses as low as 0.7mg/kg. In contrast, dexamethasone failed to induce apoptosis in the lateral septal nucleus at doses up to 20mg/kg. The levels of dexamethasone-induced striatal and hippocampal apoptosis were attenuated by pretreatment with the corticosteroid receptor antagonist RU38486 (Mifepristone), which implies that the cell death was mediated by a corticosteroid receptor-dependent process. We further determined whether dexamethasone induced sublethal damage to neurons by quantifying reductions in the number of microtubule-associated protein-2-immunoreactive striatal and hippocampal cells following injection of the corticosteroid. Dexamethasone induced dramatic decreases in the striatum, with the dorsomedial caudate-putamen being particularly affected. Similar damage was seen in the hippocampus, with the dentate gyrus and CA1 and CA3 subfields being particularly vulnerable.Equivalent corticosteroid-induced neuronal damage may occur in mood disorders, where the levels of endogenous corticosteroids are often raised. Corticosteroid-induced damage of striatal and hippocampal neurons may also account for some of the cognitive deficits seen following administration of the drugs to healthy volunteers.     

Development of full-length Trk B-immunoreactive structures in the hippocampal formation of the macaque monkey

Distribution and morphological changes of cells containing the signal transducing neurotrophin receptor, full-length Trk B (fl-Trk B), were investigated in the hippocampal formation of the macaque monkey between embryonic day 140 and the adult stage. Western blot analysis showed that one main protein band, which migrated at 141 kDa, was detected in both the embryonic and adult hippocampal formation. In the pyramidal cells in CA1 and CA3 subfields, the subiculum, and the entorhinal cortex, fl-Trk B-immunoreactive dendrites were observable in the embryonic stage. In contrast, in the granule cells of the dentate gyrus, few dendrites were immunoreactive during embryonic and early developmental stages. This difference may be due to the later growth of the granule cells of the dentate gyrus. The existence of fl-Trk B immunoreactivity in the cell body and dendrites in the embryonic hippocampal neurons, suggests that BDNF and/or NT4/5 act on the hippocampal cells by autocrine/paracrine mechanisms. In the entorhinal cortex, fl-Trk B immunoreactivity became localized in the stellate cells in layer II and the pyramidal cells in layers III, V and VI in adulthood. This indicates that BDNF and/or NT4/5 are important for the maintenance of the projection neurons in the entorhinal cortex at the adult stage. The strongest fl-Trk B immunoreactivity in the hippocampal neurons occurred at postnatal month 4, corresponding to the period of greatest synapse production in the monkey hippocampus, suggesting that BDNF and/or NT4/5 with fl-Trk B may play a role in synapse formation in the monkey hippocampus.     

GRIK4/KA1 protein expression in human brain and correlation with bipolar disorder risk variant status

The kainate class of ionotropic glutamate receptors is involved in the regulation of neuronal transmission and synaptic plasticity. Previously we reported that a deletion variant within the gene GRIK4, which encodes the KA1 kainate receptor subunit, was associated with a reduced risk of bipolar disorder and increased GRIK4 mRNA abundance. Using a high resolution immunohistochemistry technique, we characterized KA1 protein localization in human brain and performed a genotype-protein expression correlation study. KA1 was expressed in specific populations of neuronal cells in the cerebellum and all layers of the frontal and parahippocampal cortices. In the hippocampus, strong KA1 expression was observed in the stratum pyramidale and stratum lucidum of CA3 and CA2, in cell processes in CA1, in the neuropil of the CA4 region, in polymorphic cells including mossy fiber neurons in the hilus, and dentate gyrus (DG) granule cells. Mean counts of KA1 positive DG granule cells, hippocampal CA3 pyramidal cells, and layer 1 of the frontal cortex were significantly increased in subjects with the deletion allele (P?=?0.0005, 0.018, and 0.0058, respectively) compared to subjects homozygous for the insertion. Neuronal expression levels in all regions quantified were higher in the deletion group. These results support our hypothesis that the deletion allele affords protection against bipolar disorder through increased KA1 protein abundance in neuronal cells. Biological mechanisms which may contribute to this protective effect include KA1 involvement in adult hippocampal neurogenesis, HPA axis activation, or plasticity processes affecting neuronal circuitry.     

Does a unique type of CA3 pyramidal cell in primates bypass the dentate gate?

The predominant excitatory synaptic input to the hippocampus arises from entorhinal cortical axons that synapse with dentate granule cells, which in turn synapse with CA3 pyramidal cells.Thus two highly excitable brain areas--the entorhinal cortex and the CA3 field--are separated by dentate granule cells, which have been proposed to function as a gate or filter. However, unlike rats, primates have "dentate" CA3 pyramidal cells with an apical dendrite that extends into the molecular layer of the dentate gyrus, where they could receive strong, monosynaptic, excitatory synaptic input from the entorhinal cortex. To test this possibility, the dentate gyrus molecular layer was stimulated while intracellular recordings were obtained from CA3 pyramidal cells in hippocampal slices from neurologically normal macaque monkeys. Stimulus intensity of the outer molecular layer of the dentate gyrus was standardized by the threshold intensity for evoking a dentate gyrus field potential population spike. Recorded proximal CA3 pyramidal cells were labeled with biocytin, processed with diaminobenzidine for visualization, and classified according to their dendritic morphology. In response to stimulation of the dentate gyrus molecular layer, action potential thresholds were similar in proximal CA3 pyramidal cells with different dendritic morphologies. These findings do not support the hypothesis that dentate CA3 pyramidal cells receive stronger synaptic input from the entorhinal cortex than do other proximal CA3 pyramidal cells.     

Kainic acid-mediated increase of preprotachykinin-A messenger RNA expression in the rat hippocampus and a region-selective attenuation by dexamethasone.

The hippocampus contains the highest number of glucocorticoid-sensitive neurons in the rat brain and excessive exposure to glucocorticoids can cause damage to hippocampal neurons and impair the capacity of the hippocampus to survive neuronal insults. In this study in situ hybridization combined with quantitative image analysis was used to study preprotachykinin-A mRNA levels after administration of a toxic dose of kainic acid in animals pretreated with glucocorticoids. Kainic acid was injected into dorsal hippocampus CA3 region in animals pretreated with the synthetic glucocorticoid receptor agonist dexamethasone and in control animals. Preprotachykinin-A mRNA was not detected in the hippocampus of untreated animals or in animals analysed 30 min after a kainic acid injection. However, 4 h after injection of kainic acid, the level of preprotachykinin-A mRNA increased to 20-times above the detection limit both in the dentate gyrus and the CA3 region of the hippocampus. Treatment of kainic acid-injected animals with dexamethasone 30 min before and 2 h after the injection attenuated the increase in the granule cells of the dentate gyrus by 50%. In contrast, dexamethasone pretreatment had no significant effect on the kainic acid-induced increase of preprotachykinin-A mRNA in pyramidal cells in regions CA3 or CA1. These results show that an excitatory stimulus within the hippocampus causes a substantial increase in the level of preprotachykinin-A mRNA in hippocampal granule and pyramidal cells and suggest that in granule cells of the dentate gyrus this increase can be modulated by glucocorticoids.     

Septal cholinergic afferents regulate expression of brain-derived neurotrophic factor andβ-nerve growth factor mRNA in rat hippocampus

Summary In situ hybridization was used to study the expression of members of the nerve growth factor family of trophic factors in rat hippocampus following stimulation of afferent cholinergic and glutamatergic pathways with quisqualate. A transient increase in brain-derived neurotrophic factor (BDNF) and-nerve growth factor (NGF) mRNA expression in the hippocampus was seen 4 h after a quisqualate injection into the medial septal nucleus. Both BDNF and NGF mRNA levels increased more than 4-fold in the granule layer of the dentate gyrus and for BDNF mRNA also in the pyramidal cells of CA1, while the levels of BDNF mRNA in CA3 increased 2-fold. The increase in BDNF and NGF mRNA levels were completely prevented by pretreatment with systemic injections of either scopolamine or diazepam. A quisqualate injection into the entorhinal cortex, containing glutamatergic afferents to the hippocampus, resulted in a 15-, 5- and 17-fold increase in the expression of BDNF mRNA in the ipsilateral granule cells, CA3 and CA1 pyramidal cells, respectively. Similar increases were also seen in the hippocampus contralateral to the injections. In contrast, the level of NGF mRNA did not increase significantly in any of the subfields in the hippocampus. The increase in BDNF mRNA after cortex injections was attenuated by diazepam but not by scopolamine. These findings imply that increased activity in afferent cholinergic and glutamatergic pathways to the hippocampus differentially regulate expression of the NGF family of neurotrophic factors in the hippocampus.     

Regionally specific and rapid increases in brain-derived neurotrophic factor messenger RNA in the adult rat brain following seizures induced by systemic administration of kainic acid.

In situ hybridization techniques were used to analyse the spatiotemporal pattern of brain-derived neurotrophic factor messenger RNA elevation associated with kainic acid-induced seizure activity in the rat. Pronounced increases in hippocampal brain-derived neurotrophic factor messenger RNA levels were observed as early as 30 min following the onset of behavioral seizures. The greatest increase (10-fold) occurred in the dentate granule cell layer, while pyramidal layers CA1, CA3, and CA4 exhibited increases of two- to six-fold. Peak elevation of brain-derived neurotrophic factor messenger RNA in CA1 hippocampal region was evident at 4 h in CA3, and in the dentate granule layer at 30 min postseizure. Elevations persisted in the dentate and hilar regions to four days, while the increases in CA1 and CA3 returned to control levels by 16 h following seizure. Significant increases in brain-derived neurotrophic factor messenger RNA were also observed in the superficial layers of cortex (II and III) and in the piriform cortex which reached peak elevations by 8 h. No detectable changes were observed in the dorsomedial thalamus. Although histologically defined pyramidal and granule cell layers displayed relatively uniform increases in brain-derived neurotrophic factor messenger RNA in response to kainate, a closer examination of the labeling patterns using emulsion autoradiography revealed discrete areas of high grain densities overlapping uniform, moderate hybridization densities in the dentate granule cell layer and CA3, suggesting that the capacity to upregulate brain-derived neurotrophic factor messenger RNA in these regions may differ among individual neurons. In conclusion, our studies revealed that brain-derived neurotrophic factor messenger RNA induction in response to systemic kainate administration differs in hippocampal and cortical areas, in magnitude, time of onset and duration. The observed temperospatial pattern does not correspond in a simple way to increases in metabolic or electrical activity associated with seizures or neuronal vulnerability coincident with the seizures.     

不同年龄双峰驼海马的形态学变化及其神经生长因子的表达

目的 探讨胎驼、青年双峰驼及成年双峰驼海马不同区域内主要神经元形态特征变化及神经生长因子（NGF）的表达情况。 方法 应用常规石蜡切片Nissl染色及免疫组织化学方法,对胎驼、青年双峰驼和成年双峰驼海马CA1～CA3区锥体细胞、齿状回（DG)颗粒细胞的形态学变化进行研究,并观察了NGF的表达变化情况。 结果 Nissl染色结果显示,从胎驼到青年双峰驼再到成年双峰驼,海马各区锥体细胞和颗粒细胞的体积增大、密度降低、细胞的核体比减小。NGF免疫组织化学结果表明,成年双峰驼海马锥体细胞中NGF的表达量显著高于胎驼和青年双峰驼（P<0.01）,颗粒细胞中NGF的表达量逐渐增加。 结论 从胎驼到成年双峰驼,海马中的主要神经元不断成熟,从体积小且少有突起和分支的神经元发育成为体积较大、具有明显形态和功能的神经元。NGF在成年双峰驼海马神经元中表达量最高,表明NGF可调节已分化的神经元,使其形态和功能发生改变以促进成年双峰驼与海马相关的学习记忆功能的逐步完善。     

Consequences of prolonged afferent stimulation of the rat fascia dentata: epileptiform activity in area CA3 of hippocampus

Following prolonged stimulation of the perforant path input to the dentate gyrus, long-lasting changes occur in the synaptic responses and cell properties of cells in the fascia dentata. The present study describes the effects of sustained stimulation on the major population of cells innervated by the dentate granule cells: are CA3 pyramidal cells of hippocampus. In 46% of slices from rat, sustained stimulation of perforant path was followed by spontaneous, synchronized, rhythmic bursting activity in area CA3 pyramidal cells that was evident for several hours. These bursts could be recorded extracellularly in the pyramidal cell layer, throughout the hilar region, and even in the granule cell layer. With intracellular recording, all of the cells of the fascia dentata were found to be affected by the pyramidal cell bursts. Hyperpolarizing, inhibitory postsynaptic potential (IPSP)-like events occurred in all granule cells tested during the CA3 pyramidal cell burst. In contrast, spiny hilar "mossy" cells discharged synchronously with the pyramidal cells, as did some of the "fast spiking" interneurons. However, most interneurons only depolarized a few millivolts during the pyramidal cell burst. These results show that sustained stimulation of the perforant path is followed by a period of hyperexcitability in area CA3 of the hippocampus, and that hyperexcitability in area CA3 influences the activity of the cells in the fascia dentata.     

Chronic non-invasive glucocorticoid administration decreases polysialylated neural cell adhesion molecule expression in the adult rat dentate gyrus

The expression of the polysialylated neural cell adhesion molecule (PSA-NCAM) is increased in the hippocampus after chronic restraint stress (CRS) and may play a permissive role in structural changes that include dendrite reorganization in dentate gyrus (DG) and CA3 pyramidal neurons and suppression of neurogenesis in DG. We report that chronic oral corticosterone (CORT) administration decreases the number of PSA-NCAM immunoreactive granule neurons in the adult rat dentate gyrus, and the available evidence suggests that this is an indirect effect of CORT, possibly involving excitatory amino acids, that may not be directly related to neurogenesis. Because CORT treatment reduces but does not eliminate PSA-NCAM expression, the present results do not exclude a permissive role for PSA-NCAM in CORT or CRS-induced structural plasticity in hippocampus.