Distribution of glutamate receptor subunit GluR1 and GABA in human cerebral neocortex: a double immunolabelling and electron microscopic study

Specimens of human cerebral neocortex were obtained during neurosurgical operations and studied by immunocytochemistry and electron microscopy, using antibodies to the glutamate receptor subunit GluR1 and gamma-aminobutyric acid (GABA). Many GluR1-positive pyramidal neurons and fewer GluR1-positive nonpyramidal neurons were present in the cortex. Non-pyramidal neurons were more heavily labelled for GluR1 than pyramidal neurons. Most GABAergic neurons were labelled for GluR1. The white matter was unstained, except for occasional labelled neurons. This pattern of GluR1 immunostaining is similar to that in rat cerebral cortex, but is different from that in the hippocampus and amygdala, where large numbers of pyramidal or projection neurons, but few non-pyramidal or GABAergic neurons, were labelled for GluR1.     

Expression of induced nitric oxide synthase in amoeboid microglia in postnatal rats following an exposure to hypoxia

The present study showed the expression of induced nitric oxide synthase (iNOS) immunoreactivity in amoeboid microglia following an exposure to transient hypoxia in postnatal rats. iNOS immunoreactivity was expressed mainly in the amoeboid microglia in corpus callosum and subependymal regions of the ventricles within 3 h after hypoxia. The expression declined after 5 h, and became undetectable after 15 h and in longer surviving rats. The immunoreactivity of these cells with OX-42, which is a marker for microglia cells and detects complement type three receptors (CR3), was comparable in the rats exposed to hypoxia and the control rats. Immunoglobulin G (IgG) immunoreactivity was observed in the amoeboid microglia up to 3 h after hypoxia but it was undetectable in longer surviving rats and in the control rats. The iNOS expression in the amoeboid mircoglial cells may be related to the host defense and maintenance of structural integrity of the highly vulnerable periventricular white matter after hypoxia. The immunostaining of amoeboid microglial cells with IgG following hypoxia indicates leakage of plasma immunoglobulin from the blood vessels and its removal by the amoeboid microglial cells.     

Increased microglial activation and protein nitration in white matter of the aging monkey

Activated microglia are important pathological features of a variety of neurological diseases, including the normal aging process of the brain. Here, we quantified the level of microglial activation in the aging rhesus monkey using antibodies to HLA-DR and inducible nitric oxide synthase (iNOS). We observed that 3 out of 5 white matter areas but only 1 of 4 cortical gray matter regions examined showed significant increases in two measures of activated microglia with age, indicating that diffuse white matter microglial activation without significant gray matter involvement occurs with age. Substantial levels of iNOS and 3-nitrotyrosine, a marker for peroxynitrite, increased diffusely throughout subcortical white matter with age, suggesting a potential role of nitric oxide in age-related white matter injury. In addition, we found that the density of activated microglia in the subcortical white matter of the cingulate gyrus and the corpus callosum was significantly elevated with cognitive impairment in elderly monkeys. This study suggests that microglial activation increases in white matter with age and that these increases may reflect the role of activated microglia in the general pathogenesis of normal brain aging.     

An immunocytochemical study of glutamate receptors and glutamine synthetase in the hippocampus of rats injected with kainate

Immunocytochemistry was used to study the distribution of the kainate receptors GluR1, GluR2/3 and GluR4 and of the N-methyl-d-aspartate (NMDA) receptor NMDAR1 as well as the astrocyte markers glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) in the hippocampus of normal and kainate-lesioned rats. Hippocampal pyramidal neurons and dentate granule neurons were labelled heavily for GluR1 and GluR2/3, but only lightly for GluR4. Dense GluR4 immunopositivity was, however, observed in oligodendrocyte-like glial cells. Hippocampal pyramidal neurons and dentate granule neurons were moderately labelled for NMDAR1. Intravenous kainate injections resulted in a decrease in GluR1 and GluR2/3 immunoreactivity on the apical dendrites of pyramidal neurons as early as 7 h postinjection. At 18 h, there was a marked reduction in GluR1 and GluR2/3 receptors in the terminal tuft of dendrites of most hippocampal pyramidal neurons in the affected area, although some cells showed labelling in other portions of the apical dendrites and in basal dendrites. Immunostaining for GluR4 and NMDAR1 was also reduced at this time. At postinjection day 3, only the cell bodies and the basal dendrites of a few scattered pyramidal cells were labelled. Taken together, these results indicate a progressive loss of glutamate receptors, which affects the apical dendritic tree before the basal dendritic tree. The decrease in receptor immunoreactivity could be due to a downregulation of the receptors, since it occurred as early as 7 h postlesion, before cell death was evident in Nissl-stained sections. At long intervals after kainate injection, all pyramidal cells at the centre of the lesion showed a lack of glutamate receptor staining, and no partially labelled pyramidal cells were observed. The periphery of the lesion, however, contained many partially labelled pyramidal neurons among the unlabelled cells and had features of early lesions. The present study also showed an early decrease in GS immunoreactivity in the affected CA fields of the hippocampus (18 h to 3 days postinjection), followed by a medium-term increase (5–68 days) and a late decrease in GS immunoreactivity (81 days). The decrease in GS immunoreactivity at 81 days is not due to an absence of astrocytes, since GFAP staining showed many densely labelled astrocytes in the affected CA field.     

Lipid peroxidation in the postnatal rat brain

Lipid peroxidation is known to be associated with many neurodegenerative diseases and with traumatic brain injury, but its occurrence in the normal developing brain has not been reported. The present study was carried out using a specific antibody that recognises proteins modified by the end-product of lipid peroxide decomposition, 4-hydroxynonenal (HNE), to evaluate evaluate possible lipid peroxidation products in the brains of developing rats by immunocytochemistry and electron microscopy. Moderately dense labelling was observed in the supraventricular corpus callosum in the 7- and 8-day-old rats, whilst very dense labelling was observed in the same region, in the 9- and 10-day-old rats. Very little immunoreactivity was observed at 14 days, and no staining was observed in the corpus callosum in adult rats. HNE staining was not observed in neuronal cell bodies that give rise to callosal axons in the overlying cerebral cortex. Electron microscopy showed dense HNE staining on the basal laminae of blood vessels and on the plasma membranes of unmyelinated axons. Large numbers of rounded cells with features of oligodendrocyte precursor cells were labelled by Perl's stain in the supraventricular corpus callosum at postnatal day 7 and postnatal day 10, i.e. at times corresponding to high levels of HNE immunoreactivity. In contrast, very few such cells were observed in the adult brain, corresponding to the very little or no Perl's staining in the adult. These results suggest that lipid peroxidation observed in the supraventricular corpus callosum at postnatal day 10 could result from an accumulation of iron in this region, at this time.     

Immunocytochemical localization of CR3 complement receptors with OX-42 in amoeboid microglia in postnatal rats

Summary The present study described the labelling of amoeboid microglial cells in the postnatal rat brain with OX-42, an antibody that recognizes type 3 complement receptors CR3 in mononuclear phagocytes. Of the diverse morphological forms of amoeboid microglia present in the corpus callosum in early postnatal (2–5 days) rats, cells with a round regular outline, or showing short stout processes, were the most intensely stained. When traced from the main cell colony into the borderline zone with the cortex, the immunoreactivity of amoeboid microglia that assumed a ramified form was drastically reduced. Examination of materials from the late postnatal (8–12 days) age group showed that the majority of the OX-42 positive cells in the corpus callosum became oval, elongated and ramified. Immunoelectron microscopy confirmed the above observations, and also showed that the immunoreactivity in the round amoeboid microglia was localized in their plasma membrane, surface projections and invaginations, as well as in some of the subsurface vacuoles. The immunoreactivity was reduced in the oval cells, and diminished in the elongated or ramified form. It is proposed that the presence of CR3 membrane receptors in amoeboid microglial cells is related to their active role in endocytosis. These, however, diminish with the growth of the brain.     

Neuronal ectopic masses induced by prenatal irradiation in the rat

Summary Ectopic neuronal masses below the subcortical white matter were seen in the brains of postnatal rats after 200 cGy irradiation at embryonic day 14. In contrast with the laminated organisation of the cortex located above the subcortical white matter, the ectopic masses were formed of confluent nodules composed of pyramidal and non-pyramidal neurons distributed at random, with no laminar organisation. Afferent and efferent fibres to/from the ectopic masses running together with fibres passing the subcortical white matter indicated that the ectopic masses were heavily connected to neighbouring structures. Examination of irradiated embryos revealed that the ectopic masses originated from ectopic periventricular rosettes, composed of germinal cells, which were formed shortly after irradiation. Neuronogenesis in these rosettes did not follow an inside-out gradient, as seen in the laminated cortex; however, early-generated neurons predominated in the external regions, whereas late-generated neurons were mainly located in the middle and internal regions of the ectopic masses.     

The relationship between callosal axons and cortical neurons in the planum temporale: alterations in schizophrenia

The relationship between “connectivity” measures such as DTI and the cellular alterations in the cortex that give rise to those connections remains unclear. Cytoarchitectural changes in the planum temporale (PT) suggest impaired layer III feedforward projection neurons in schizophrenia. Altered hemispheric asymmetry of the PT has been reported in patients, along with altered white matter density in the corpus callosum, and there is anomalous activation of the PT during auditory hallucinations. We measured layer III cell density and pyramidal neuron size in PT of both hemispheres of post-mortem brains from patients with schizophrenia (n = 16) and control subjects (n = 16). We found reduced cell density and the loss of a correlation between magnopyramidal neuron density and axon number in the isthmus of the corpus callosum in schizophrenia. The normal asymmetry indicated that magnopyramidal neurons tend towards being larger and denser in the left PT but this asymmetry is significantly reduced in schizophrenia. The findings offer cytoarchitectural insight into the relationship between PT cortex and callosal white matter abnormalities in schizophrenia.     

胚胎期X线照射致大鼠皮质下异位细胞团形成和胼胝体缺失的相关性研究

为探讨胚胎期大鼠受X线照射后皮质下异位细胞团形成与胼胝体缺失的相关性,本研究将妊娠14d(E14)大鼠经X线全身照射(剂量为1.0Gy),在照射前2h及照射后2d(E16)经尾静脉注入5-溴-2-脱氧尿嘧啶(BrdU),用以标识E14及E16的神经细胞的发生。取不同时期(E14～P3)胚脑用于Nissl染色及免疫组化染色分析。结果显示:(1)胚胎期经X线照射的新生大鼠可见新皮质变薄,胼胝体缺失,以及皮质板下的异位细胞团形成;(2)在X线照射前2h注射BrdU:大鼠经照射后6h(E14～6h),侧脑室内可见大量BrdU阳性细胞;在E16和E18时,BrdU阳性细胞表达于皮质边缘带及下板层,但数目远少于未接受X线照射的对照组;(3)X线照射后2d即E16时注入BrdU:大鼠在E20和P1时,BrdU阳性细胞与对照组同样分别表达于皮质板的上层及下层,但数目较对照组明显减少;同时可见大脑皮质第II、III层细胞大部分缺损以及由BrdU弱阳性细胞聚集在皮质板最下层形成的异位细胞团。本实验结果证明:X线照射可损伤多数正处于分裂期的神经上皮细胞,且后续发生的部分神经细胞不能正常移动到他们的目的地(皮质板第II、III层)而聚集在皮质板下成为异位细胞团。由于胼胝体主要由大脑皮质第II、III层细胞的轴突构成,我们推测异位细胞团的形成可导致胼胝体的缺失。     

The cuprizone model: regional heterogeneity of pathology

The cuprizone model is a model of de- and remyelination secondary to oligodendrocyte death, likely to be mediated by an inhibition of mitochondrial function. The aim of this study was to characterize histopathological changes associated with de/remyelination in grey and white matter at different disease stages in C57Bl/6 mice after per oral administration of cuprizone. Oligodendrocyte loss, astrocytosis and complement activation was detected in areas of demyelination. Demyelination, astrocytosis and complement activation occurred earlier in the cerebral cortex than in the corpus callosum. There was no perivascular lymphocyte infiltration. Microglia- and macrophage activation was observed in the corpus callosum, but not in the cerebral cortex. After cuprizone exposure was stopped, remyelination was extensive in the corpus callosum, but scarce in the cortex. In conclusion, cortical demyelination and oligodendrocyte loss in the cuprizone model may be due to a direct effect on oligodendrocyte mitochondrial function, as it occurs in the absence of microglial activation. The histopathology of de/remyelination in the cuprizone treated mice show regional heterogeneities which suggest differences in the underlying pathophysiology. Cuprizone-induced demyelination is a relevant model for the study of regional heterogeneity of demyelination and lesion pathology in multiple sclerosis.     

Thalamic inputs to identified commissural neurons in the monkey somatic sensory cortex

Summary Commissurally projecting neurons were identified in the monkey first somatic sensory area (SI) by the retrograde axonal transport of horseradish peroxidase (HRP) injected into the contralateral cortex. Neurons identified in this way have large pyramidal somata primarily in layer IIIB of the SI area. Their basal dendrites lie within the terminal plexus of thalamocortical afferents.Electron microscopy was used to examine the synaptic relations of the labelled commissural cells, in particular to determine whether they receive monosynaptic thalamic connections. To do this, retrogradely labelled commissural cells and Golgi-impregnated large pyramidal neurons from layer IIIB were examined ultrastructurally in material in which thalamocortical terminals were degenerating due to a prior lesion of the thalamus. In a significant number of cases degenerating terminals were found to make synapses on the spines or shafts of labelled dendrites.Injections of HRP into SI or into the white matter adjacent to the corpus callosum labelled callosal axons and terminals in the opposite SI. These axons terminated mainly near the somata of the layer IIIB pyramidal cells. Some of their terminals were found to synapse with dendrites receiving synaptic contacts from thalamocortical axon terminals.     

Differential localisation of the metabotropic glutamate receptor mGluR1a and the ionotropic glutamate receptor GluR2/3 in neurons of the human cerebral cortex

Specimens of human cerebral cortex were obtained during neurosurgical operations and studied by immunocytochemistry and electron microscopy, using antibodies to the metabotropic glutamate receptor subunit mGluR1a and the ionotropic glutamate receptor GluR2/3. A small number of non-pyramidal neuronal cell bodies were labelled for mGluR1a. Double immunolabelling with mGluR1a and GluR2/3 showed that most pyramidal cell bodies were labelled for GluR2/3 but not for mGluR1a. Despite the non-colocalisation of these two receptor subtypes in cell bodies, however, many dendrites and dendritic spines were double-labelled for mGluR1a and GluR2/3 at electron microscopy. As there is evidence that most neurons positive for GluR2/3 are pyramidal cells, this suggests that mGluR1a is present in dendrites of pyramidal neurons, despite absent or low levels of immunoreactivity in their cell bodies. Received: 5 May 1997 / Accepted: 24 July 1997     

Populations of subplate and interstitial neurons in fetal and adult human telencephalon

In the adult human telencephalon, subcortical (gyral) white matter contains a special population of interstitial neurons considered to be surviving descendants of fetal subplate neurons [Kostovic & Rakic (1980) Cytology and the time of origin of interstitial neurons in the white matter in infant and adult human and monkey telencephalon. J Neurocytol9, 219]. We designate this population of cells as superficial (gyral) interstitial neurons and describe their morphology and distribution in the postnatal and adult human cerebrum. Human fetal subplate neurons cannot be regarded as interstitial, because the subplate zone is an essential part of the fetal cortex, the major site of synaptogenesis and the 'waiting' compartment for growing cortical afferents, and contains both projection neurons and interneurons with distinct input-output connectivity. However, although the subplate zone is a transient fetal structure, many subplate neurons survive postnatally as superficial (gyral) interstitial neurons. The fetal white matter is represented by the intermediate zone and well-defined deep periventricular tracts of growing axons, such as the corpus callosum, anterior commissure, internal and external capsule, and the fountainhead of the corona radiata. These tracts gradually occupy the territory of transient fetal subventricular and ventricular zones.The human fetal white matter also contains distinct populations of deep fetal interstitial neurons, which, by virtue of their location, morphology, molecular phenotypes and advanced level of dendritic maturation, remain distinct from subplate neurons and neurons in adjacent structures (e.g. basal ganglia, basal forebrain). We describe the morphological, histochemical (nicotinamide-adenine dinucleotide phosphate-diaphorase) and immunocytochemical (neuron-specific nuclear protein, microtubule-associated protein-2, calbindin, calretinin, neuropeptide Y) features of both deep fetal interstitial neurons and deep (periventricular) interstitial neurons in the postnatal and adult deep cerebral white matter (i.e. corpus callosum, anterior commissure, internal and external capsule and the corona radiata/centrum semiovale). Although these deep interstitial neurons are poorly developed or absent in the brains of rodents, they represent a prominent feature of the significantly enlarged white matter of human and non-human primate brains.     

The relationship between callosal axons and cortical neurons in the planum temporale: Alterations in schizophrenia

The relationship between “connectivity” measures such as DTI and the cellular alterations in the cortex that give rise to those connections remains unclear. Cytoarchitectural changes in the planum temporale (PT) suggest impaired layer III feedforward projection neurons in schizophrenia. Altered hemispheric asymmetry of the PT has been reported in patients, along with altered white matter density in the corpus callosum, and there is anomalous activation of the PT during auditory hallucinations. We measured layer III cell density and pyramidal neuron size in PT of both hemispheres of post-mortem brains from patients with schizophrenia (n = 16) and control subjects (n = 16). We found reduced cell density and the loss of a correlation between magnopyramidal neuron density and axon number in the isthmus of the corpus callosum in schizophrenia. The normal asymmetry indicated that magnopyramidal neurons tend towards being larger and denser in the left PT but this asymmetry is significantly reduced in schizophrenia. The findings offer cytoarchitectural insight into the relationship between PT cortex and callosal white matter abnormalities in schizophrenia.     

Response of amoeboid and differentiating ramified microglia to glucocorticoids in postnatal rats: a lectin histochemical and ultrastructural study.

After glucocorticoid injection(s), the number of amoeboid microglial cells (AMC) in the corpus callosum labelled by lectin was markedly reduced when compared with the corresponding control rats. In rats killed at the age of 7 days, all the labeled cells differentiated to become ramified microglia. Ultrastructurally, the AMC in glucocorticoid-injected rats were extremely vacuolated and showed increased lipid droplets. Furthermore, the cells displayed varied lectin labelling patterns especially at both the trans saccules of the Golgi apparatus and lysosomes. In differentiating ramified microglia, massive cellular debris and lectin-stained vesicles or vacuoles were observed; some of the latter appeared to fuse with the plasma membrane. The most striking feature after glucocorticoid (GCC) treatment was the complete diminution of lectin labelling at the Golgi saccules in some differentiating ramified microglia. The present results have demonstrated different effects of glucocorticoids on AMC and differentiating ramified microglia. The differential response of AMC and differentiating ramified microglia to the immunosuppressive drugs may be attributed to the fact that these cells in the postnatal brains subserve different functions or that they are at different differentiation stages. In other words, the sensitivity of microglial cells to the immunosuppressive drugs is dependent upon the stage of cell maturation/differentiation.     

On the projections from the neostriatum to the cerebral cortex: the "displaced" neurons

The entire dorsal and lateral cortex of one cerebral hemisphere of rats was infiltrated with different fluorescent tracers and the neostriatum was examined for labelled perikarya. In spite of the extensive infiltration of the cortex, such neurons were seen only sporadically in the ipsilateral neostriatum and almost only in the vicinity of the globus pallidus and the subcortical white matter. The size and shape of these neurons, and particularly their proximity to some cell groups which surround the neostriatum and project to the cerebral cortex, suggest that these neurons belong to the neighbouring structures such as the magnocellular nuclei of the basal forebrain, the claustrum and the VIb cortical layer.     

Acute Marchiafava-Bignami disease with widespread callosal and cortical lesions

Marchiafava-Bignami disease (MBD) is a rare alcohol-related disorder that results in progressive demyelination and necrosis of the corpus callosum. The process may extend to the optic chiasm and tracts, cerebellar peduncle, subcortical resion, neighboring white matter, and rarely, cortical gray matter. We report a case of MBD in which fluid-attenuated inversion recovery and diffusion magnetic resonance imaging studies revealed symmetrical hyperintense lesions in the cerebral cortex in addition to the callosal lesions.     

The inhibitory effect of the ventrolateral periaqueductal grey matter on neurones in the rostral ventrolateral medulla involves a relay in the medullary raphe nuclei

We have investigated the expression of messenger RNA (mRNA) encoding substance P receptor (SPR) in the visual cortex of adult cats and 17-day-old kittens, using in situ hybridization histochemistry with two digoxigenin-labeled oligodeoxynucleotides complementary to the SPR mRNA. In the adult cortex, a subset of large pyramidal neurons of layer V and layer III is heavily labeled. Other, mainly pyramidal neurons in layers II, III and V are less intensely labeled, but most neurons in these layers appear unlabeled. Neurons in layer IV and VI, and in the white matter do not show hybridization signals above background levels. In the 17-day-old kitten, SPR mRNA-expressing cells are confined to layer V and to the upper white matter (subplate zone), whereas supragranular neurons do not yet contain SPR mRNA. A few neurons in layer VI display moderate labeling. Astrocytes, identified with anti-glial fibrillary acid protein antibodies, did not express detectable levels of SPR mRNA in both adult and kitten visual cortex. These results indicate that SPR mRNA expression is transient in neurons of the white matter, and is developmentally regulated in supragranular layers. In addition, the localization of SPR mRNA in a subset of pyramidal cells suggests that substance P modulates the excitability of certain projection neurons which are the origin of extrinsic connections.     

Experimentally induced laminar necrosis,status verrucosus,focal cortical dysplasia reminiscent of microgyria,and porencephaly in the rat

Different types of cortical malformation were produced, following focal cortical freezing, electrocoagulation, focal cortical aspiration or gentle brushing of uncovered meninges, in newborn or 1-to 3-day-old rats. Malformations included laminar necrosis of the cerebral cortex, status verrucosus, focal cortical dysplasia reminiscent of microgyria, and porencephaly. Similar procedures from postnatal day 4 onwards, at a time when a reactive astrogliosis is possible, produced cavitating infarcts and tissue scars. Cytoarchitectonic studies revealed an abnormal distribution of different types of pyramidal and nonpyramidal neurons in these malformations. These indicated three subtypes of focal cortical dysplasia, which probably depend on different pathogenic mechanisms. Autoradiographic studies with [³H] methylthymidine showed normal positioning of late-generated neuroblasts in the cerebral cortex, thus suggesting preserved migration. The present experimentally induced cortical malformations are useful models of similar cortical abnormalities in humans.     

天麻素对缺血缺氧脑损伤新生大鼠小胶质细胞Sirt3表达的影响

目的:研究天麻素对缺血缺氧脑损伤(HIBD)新生大鼠脑内激活的小胶质细胞Sirt3表达的影响。方法:选取39只3 d龄SD幼年大鼠随机分为对照组(control)、缺血缺氧脑损伤组(HIBD)、天麻素组(HIBD+gastrodin)。采用左侧颈总动脉结扎结合缺氧法建立新生大鼠缺血缺氧性脑损伤(HIBD)模型,使用免疫荧光染色观察HIBD模型后新生大鼠大脑胼胝体区小胶质细胞的分布情况;使用免疫荧光双标染色及Western Blot检测大鼠大脑左侧胼胝体区小胶质细胞Sirt3的表达变化。结果:免疫荧光染色结果显示HIBD后新生大鼠大脑左侧胼胝体区小胶质细胞出现明显激活,确定模型建立成功。免疫荧光双标染色及Western Blot结果均显示,与对照组相比,HIBD模型组Sirt3的表达水平明显升高(P <0. 05);与HIBD模型组相比,天麻素组Sirt3的表达进一步增强(P <0. 05)。结论:天麻素可能通过促进新生大鼠脑内小胶质细胞Sirt3的表达,发挥其神经保护作用。