Spatiotemporal Profile and Morphological Changes of NG2 Glia in the CA1 Region of the Rat Hippocampus after Transient Forebrain Ischemia

Neuron-glial antigen-2 (NG2) glia undergo proliferation and morphological changes following brain insults. Here, we show that NG2 glia is activated in a characteristic time- and layer-specific manner in the ischemia-vulnerable CA1 region of the rat hippocampus. Resting NG2 glia of the pyramidal cell layer (somatic region) shared morphological features with those of the neighboring dendritic stratum radiatum. During the postischemic period, reactive NG2 glia of the pyramidal cell layer exhibited shortened, scarcely branched processes, while those of the stratum radiatum had multiple branching processes with their arborization being almost indiscernible 7~14 days after reperfusion. Immunoelectron microscopy demonstrated that NG2 immunoreactivity was specifically associated with the plasma membrane and the adjacent extracellular matrix of NG2 glia in the stratum radiatum at 14 days. NG2 glia also exhibited differences in their numbers and proliferation profiles in the two examined hippocampal strata after ischemia. In addition, induced NG2 expression in activated microglia/macrophages exhibited a characteristic strata-dependent pattern in the ischemic CA1 hippocampus. NG2 induction was prominent in macrophage-like phenotypes which were predominantly localized in the pyramidal cell layer, compared with activated stellate microglial cells in the stratum radiatum. Thus, our data demonstrate that activation of NG2 glia and the induction of NG2 expression in activated microglia/macrophages occur in a distinct time- and layer-specific manner in the ischemic CA1 hippocampus. These characteristic profiles of reactive NG2 glia could be secondary to the degeneration processes occurring in the cell bodies or dendritic domains of hippocampal CA1 pyramidal neurons after ischemic insults.

Increased expression of vascular endothelial growth factor-C and vascular endothelial growth factor receptor-3 after pilocarpine-induced status epilepticus in mice

Vascular endothelial growth factor (VEGF)-C and its receptor, vascular endothelial growth factor receptor (VEGFR)-3, are responsible for lymphangiogenesis in both embryos and adults. In epilepsy, the expression of VEGF-C and VEGFR-3 was significantly upregulated in the human brains affected with temporal lobe epilepsy. Moreover, pharmacologic inhibition of VEGF receptors after acute seizures could suppress the generation of spontaneous recurrent seizures, suggesting a critical role of VEGF-related signaling in epilepsy. Therefore, in the present study, the spatiotemporal expression of VEGF-C and VEGFR-3 against pilocarpine-induced status epilepticus (SE) was investigated in C57BL/6N mice using immunohistochemistry. At 1 day after SE, hippocampal astrocytes and microglia were activated. Pyramidal neuronal death was observed at 4 days after SE. In the subpyramidal zone, VEGF-C expression gradually increased and peaked at 7 days after SE, while VEGFR-3 was significantly upregulated at 4 days after SE and began to decrease at 7 days after SE. Most VEGF-C/VEGFR-3-expressing cells were pyramidal neurons, but VEGF-C was also observed in some astrocytes in sham-manipulated animals. However, at 4 days and 7 days after SE, both VEGFR-3 and VEGF-C immunoreactivities were observed mainly in astrocytes and in some microglia of the stratum radiatum and lacunosum-moleculare of the hippocampus, respectively. These data indicate that VEGF-C and VEGFR-3 can be upregulated in hippocampal astrocytes and microglia after pilocarpine-induced SE, providing basic information about VEGF-C and VEGFR-3 expression patterns following acute seizures.

Characterization of nestin expression in astrocytes in the rat hippocampal CA1 region following transient forebrain ischemia / 대한해부학회지

Recent studies have suggested that nestin facilitates cellular structural remodeling in vasculature-associated cells in response to ischemic injury. The current study was designed to investigate the potential role of post-ischemic nestin expression in parenchymal astrocytes. With this aim, we characterized ischemia-induced nestin expression in the CA1 hippocampal region, an area that undergoes a delayed neuronal death, followed by a lack of neuronal generation after transient forebrain ischemia. Virtually all of the nestin-positive cells in the ischemic CA1 hippocampus were reactive astrocytes. However, induction of nestin expression did not correlate simply with astrogliosis, but rather showed characteristic time- and strata-dependent expression patterns. Nestin induction in astrocytes of the pyramidal cell layer was rapid and transient, while a long-lasting induction of nestin was observed in astrocytes located in the CA1 dendritic subfields, such as the stratum oriens and radiatum, until at least day 28 after ischemia. There was no detectable expression in the stratum lacunosum moleculare despite the evident astroglial reaction. Almost all of the nestin-positive cells also expressed a transcription factor for neural/glial progenitors, i.e., Sox-2 or Sox-9, and some cells were also positive for Ki-67. However, all of the nestin-positive astrocytes expressed the calcium-binding protein S100beta, which is known to be expressed in a distinct, post-mitotic astrocyte population. Thus, our data indicate that in the ischemic CA1 hippocampus, nestin expression was induced in astroglia that were becoming reactive, but not in a progenitor/stem cell population, suggesting that nestin may allow for the structural remodeling of these cells in response to ischemic injury.

Influence of Aspirin on Pilocarpine-Induced Epilepsy in Mice

Aspirin (acetylsalicylic acid) is one of the most widely used therapeutic agents based on its pharmacological actions, including anti-inflammatory, analgesic, anti-pyretic, and anti-thrombotic effects. In this study, we investigated the effects of aspirin on seizure susceptibility and hippocampal neuropathology following pilocarpine-induced status epilepticus (SE). SE was induced by pilocarpine hydrochloride (280 mg/kg, i.p.) administration in C57BL/6 mice (aged 8 weeks). Aspirin was administered daily (15 mg/kg or 150 mg/kg, i.p.) for 10 days starting 3 days before SE, continuing until 6 days after SE. After pilocarpine injection, SE onset time and mortality were recorded. Neuronal cell death was examined using cresyl violet and Fluoro-Jade staining, and glial responses were observed 7 days post SE using immunohistochemistry. In the aspirin-treated group, the onset time of SE was significantly shortened and mortality was markedly increased compared to the control group. However, in this study, aspirin treatment did not affect SE-induced neuronal cell death or astroglial and microglial responses in the hippocampus. In conclusion, these results suggest that the safety of aspirin should be reevaluated in some patients, especially with neurological disorders such as temporal lobe epilepsy.

Development of Human Neuro-digital Slides and Neuro-atlas for Neuroscience Tutorial I (Spinal Cord and Brain Stem) / 체질인류학회지

In order to present the optimal neuroscience tutorial material for medical students and researchers, this study is aimed to make neuro-digital slide and neuro-atlas based on the histological specimens of human spinal cord and brain stem. Cadavers which had agreed for organ donation for research purpose were used in this study. Brains and spinal cords were extracted within 24 hours after death, and then fixed with 10% neutral buffered formalin. Paraffin blocks were made with the following regions; 8 regions from the spinal cord (the levels of the upper cervical segment, the cervical enlargement, the upper thoracic segment, the mid thoracic segment, the lower thoracic segment, the upper lumbar segment, the lumbar enlargement, the sacral segment), 14 regions from the brain stem (the levels of the spinomedullary junction, the pyramidal decussation, the medial lemniscus decussation, the obex, the mid-olivary medulla, the upper medulla, the pontomedullary junction, the lower pons, the mid pons, the upper pons, the isthmus rhombencephali, the inferior colliculus, the superior colliculus, the posterior commissure). Using virtual microscope software, we made digital neuro-slides which can be used anywhere and anytime regardless of equipment of microscope. To help understanding anatomy and functions of nervous tissue, we also made neuro-atlas based on the digital slide images. As results, the outline and detailed structures of nuclei and tracts are easily discriminated and also matched with marks and nomenclatures of neuro-atlas. Moreover, the cytoarchitecture of each nucleus and histological features can be well distinguished. We hope that this product would be used as a useful neuroscience tutorial material for the medical and paramedical school students, clinical trainees like interns and residents, and also neuroscience researchers.

Bis Is Involved in Glial Differentiation of P19 Cells Induced by Retinoic Acid

Previous observations suggest that Bis, a Bcl-2-binding protein, may play a role the neuronal and glial differentiation in vivo. To examine this further, we investigated Bis expression during the in vitro differentiation of P19 embryonic carcinoma cells induced by retinoic acid (RA). Western blotting and RT-PCR assays showed that Bis expression was temporarily decreased during the free floating stage and then began to increase on day 6 after the induction of differentiation. Double immunostaining indicated that Bis-expressing cells do not express several markers of differentiation, including NeuN, MAP-2 and Tuj-1. However, some of the Bis-expressing cells also were stained with GFAP-antibodies, indicating that Bis is involved glial differentiation. Using an shRNA strategy, we developed bis-knock down P19 cells and compared them with control P19 cells for the expression of NeuroD, Mash-1 and GFAP during RA-induced differentiation. Among these, only GFAP induction was significantly attenuated in P19-dnbis cells and the population showing GFAP immunoreactivity was also decreased. It is noteworthy that distribution of mature neurons and migrating neurons was disorganized, and the close association of migrating neuroblasts with astrocytes was not observed in P19-dnbis cells. These results suggest that Bis is involved in the migration-inducing activity of glial cells.

Effects of Apigenin on Glutamate-induced Ca2+i Increases in Cultured Rat Hippocampal Neurons

Flavonoids have been shown to affect calcium signaling in neurons. However, there are no reports on the effect of apigenin on glutamate-induced calcium signaling in neurons. We investigated whether apigenin affects glutamate-induced increase of free intracellular Ca2+concentration ([Ca2+]i) in cultured rat hippocampal neurons, using fura-2-based digital calcium imaging and microfluorimetry. The hippocampal neurons were used between 10 and 13 days in culture from embryonic day 18 rats. Pretreatment of the cells with apigenin (1micrometerto 100micrometer for 5 min inhibited glutamate (100 micrometer 1 min) induced [Ca2+]i increase, concentration-dependently. Pretreatment with apigenin (30micrometer for 5 min significantly decreased the [Ca2+]i responses induced by two ionotropic glutamate receptor agonists, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA, 10 micrometer 1 min) and N-methyl-D-aspartate (NMDA, 100 micrometer 1 min), and significantly inhibited the AMPA-induced peak currents. Treatment with apigenin also significantly inhibited the [Ca2+]i response induced by 50 mM KCl solution, decreased the [Ca2+]i responses induced by the metabotropic glutamate receptor agonist, (S)-3,5-dihydroxyphenylglycine (DHPG, 100micrometer 90 s), and inhibited the caffeine (10 mM, 2 min)-induced [Ca2+]i responses. Furthermore, treatment with apigenin (30micrometer significantly inhibited the amplitude and frequency of 0.1 mM [Mg2+o-induced [Ca2+]i spikes. These data together suggest that apigenin inhibits glutamate-induced calcium signaling in cultured rat hippocampal neurons.

Effect of Vascular Endothelial Growth Factor (VEGF) on Neuronal and Glial Response Following Transient Global Ischemia in Rats

OBJECTIVE: The authors present the effect of VEGF upon neuronal and glial response following transient global ischemia of the rat METHODS: We studied the effect of VEGF in 36 rats subjected to 15 minutes of transient global ischemia. Animals were devided into control group(transient global ischemia only: day-3, day-7, day-14, respectively n=6) and VEGF-treated group(transient global ischemia with intraventricular injection of 100 micro gram VEGF: day-3, day-7, day-14, respectively n=6). These animals were sacrificed at 3 days, 7 days and 14 days after induction of ischemia. Nissle stain and immunohistochemistry of GFAP(glial fibrillary acidic protein), OX-42, and ED1 were done for assessment of neuronal and glial responses. RESULTS: In the CA1 hippocampus, there was a significant reduction of pyramidal cell damage in VEGF-treated group as compared with control group in post-ischemia 3, 7, 14 days(p0.05). In the assessment of CA1 hippocampus with GFAP stained areas, there was significant reduction of reactivity in post-ischemia 3, 7 days(p0.05). In the CA3 hippocampus, reduction of GFAP reactivity was significant in post-ischemia 3, 7 days(p0.05). In the assessment of CA1 hippocampus with OX-42 stained areas, there was significant reduction of reactivity in post-ischemia 3, 7, 14 days(p<0.05). But in the CA3 hippocampus, the difference was not significant in post-ischemia 3, 7 days(p<0.05). In the assessment of of CA1 hippocampus with ED1 stained areas, there was significant reduction of reactivity in post-ischemia 3, 7, 14 days(p<0.05). But in the CA3 hippocampus, the difference was significant in post-ischemia 3 days only(p<0.05). CONCLUSION: These results suggest that VEGF can reduce neuronal damage in transient global ischemia, thus have protective effect on ischemic brain injury. In our experiment, the reduction of glial response with VEGF seems to be related to the secondary neuroprotective effect of VEGF. However, the proliferation of endothelial cells and new vessel formation take days to months, the thus neuroprotective effect of VEGF against ischemia seems to related to a certain mechanism rather than angiogenesis.

Induction of Bis, a Bcl-2-binding protein, in reactive astrocytes of the rat hippocampus following kainic acid-induced seizure

The expression of Bis (also called Bag-3), a Bcl-2-binding protein, was investigated in the rat kainic acid (KA) model of temporal lobe epilepsy. Western blot analysis showed a significant increase in the expression levels of Bis protein in the hippocampus following the systemic administration of KA. Bis immunoreactivity increased preferentially in the CA1 and CA3 regions, as well as in the hilar region of the dentate gyrus. Experiments with double immunofluorescence revealed that, in KA-administered rats, the cells expressing Bis were GFAP-expressing reactive astrocytes. The increase in Bis immunoreactivity was accompanied by increased Bcl-2 in reactive astrocytes in the striatum radiatum, whereas Bcl-2 immunoreactivity in pyramidal neurons was not affected. These results of the co-expression of Bis and Bcl-2 in reactive astrocytes in this seizure model suggest that Bis might modulate the glial reaction under excitotoxic brain injury, probably by interacting with Bcl-2.

Immunohistochemical Localization of Phospholipase D1 in Developing Rat Forebrain / 대한해부학회지

Phospholipase D (PLD), one of the intracellular signal transduction enzymes, may play an important role in developing brain. However, the developmental regulation of PLD protein has not been determined. In the present study, we investigated the temporal and spatial expression of PLD isozyme, PLD1 in the developing rat forebrain using an affinity-purified peptide antibody against PLD1. Our data showed that immunoreactivity for PLD1 was first seen in the germinal zone of the lateral ventricle, differentiating neurons and their processes at embryonic day 18 (E18). At E20, clusters of immunoreactive cells were observed in the medial germinal zone of the lateral ventricle, restricted zones of the frontal and parietal cortex, the nuclei of the medial septum and the diagonal band. During the first postnatal week, there was an increase in the number and staining intensity of the immunoreactive neurons in the cerebral cortex, which peaked at postnatal day 7 (P7). During the second postnatal week, there was an abrupt decrease in the number of immunoreactive cortical pyramidal neurons. By P14, only a few of the pyramidal neurons in cerebral cortex layer V were immunoreactive. These results revealed that expression of PLD1 protein at various stages of development of the septum and cerebral cortex is differentially regulated. This suggests that PLD1 may regulate the developmental processes of some neuronal populations.

Differential Expression of GABA Transporters in Developing Rat Retina / 대한해부학회지

To investigate the spatial and temporal distribution of g-aminobutyric acid (GABA) transporters in the developing rat retina, we localized two GABA transporter proteins, GAT-1 and GAT-3 by immunocytochemistry. GAT-1 immunoreactivity appeared from embryonic day 20 (E20) in the punctate-like structures in the inner plexiform layer. At postnatal day 3 (P3), immunolabeling of cell bodies in the inner nuclear and ganglion cell layers and processes in the inner plexiform layer became much stronger, reaching a maximum staining intensity during the second postnatal week, and the expression pattern and intensity became almost identical to those of the adult retina by P14. In addition, Miller cells also began to show weak immunlabeling for GAT-1 from P10 onward. In contrast, develop-mentally transient immunolabeling for GAT-1 was found in horizontal cells located at the scleral border of the inner nuclear layer during the second postnatal week. The initial immunolabeling for GAT-3 immunoreactivity was already noted in scattered cell bodies and processes of the neuroblastic layer at E18, the earliest time point. During the first week of the postnatal life, GAT-3 immunoreactivity increased in the cell bodies in the inner nuclear and ganglion cell layers, and processes in the inner plexiform layer. From P10 onward, this labeling began to decline, and remained faintly in the neuronal somata of the inner nuclear layer by P14. Instead, the labeling was predominantly localized in Mler cells. Astrocytes in the nerve fiber layer showed the transient labeling during the first postnatal week. Our results showed distinct temporal patterns of two GABA transporter proteins in the developing rat retina, and it suggests specialized roles for GABA transporters in the development of the rat retina.

Expression of GABA Transporters in the Rat's Olfactory Bulb during Development

Developmental changes in the expression of two GABA (gamma-aminobutyric acid) transporter proteins, GAT-1 and GAT-3, in the olfactory bulb of embryonic and postnatal rats were examined with immunocytochemistry using antisera against GAT-1 and GAT-3. The expression and localization of GAT-1 and GAT-3 showed distinct temporal patterns during olfactory bulb development. GAT-1 immunoreactivity appeared weakly in most likely growing axons of the presumptive glomerular layer from embryonic day 18 and increased during the first postnatal week. In contrast, GAT-3 immunoreactivity, first detected at E16, was found in radial glial cell fascicles and was replaced by what were likely astroglial cells postnatally. At P7, GAT-1 and GAT-3 immunoreactivities reached the adult pattern i.e., GAT-1 immunoreactivity was observed in the labeled punctate structures in all layers of the olfactory bulb except the nerve fiber layer, while GAT-3 immunoreactivity was observed in the astroglial processes of all layers of the olfactory bulb. Our results suggest that GABA transporters, especially GAT-3, play important roles in regulating the GABA levels of developing olfactory bulbs.

Expression of Nitric Oxide Synthase in the Olfactory Bulb of the Rat during Development / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The presence and distribution of NADPH-diaphorase activity in the olfactory bulb during development has been reported. But the precise localization of NO-synthase (NOS) in the olfactory bulb during the developmental stages has not been studied yet. Therefore, we investigated the localization of NOS-immunoreactivity in a developing rat olfactory bulb by immunohistochemistry. MATERIALS AND METHODS: A total of 32 male and female Sprague-Dawley rats. They were of several prenatal and postnatal stages, such as the following: embryonic day 16 (E16), E18, E20, postnatal day 1 (P1), P5, P7, P14 and adult. Indirect immunoperoxidase method using rabbit polyclonal anti-bNOS antibody was performed for detecting the NOS immunoreactivity. RESULTS: In the main olfactory bulb, the first NOS-immunoreactive (IR) neurons were observed in the presumptive granule cell layer (GCL) by E18, and in the glomerular layer (GL) by P1. The density of these neurons was increased as the development stage approached the adult stage. In the GCL, two types of NOS-immunoreactive neurons were observed: intensively stained large, short axon cells and weakly stained small, granule cells. The first, localized in the deeper part of the GCL, was observed in the earlier developmental stages, and the latter which increased in number to the adult period was observed by P1. In the accessory olfactory bulb, NOS-IR neurons were first detected in the GCL by P1, and increased in number to the adult period. The pattern of NOS-IR neurons in the GCL of the accessory olfactory bulb is similar to that in the main olfactory bulb. CONCLUSION: Our results demonstrated that bNOS had a characteristic temporal and spatial patterns of expression in the main and accessory olfactory bulb of the rat during development.

Distribution Pattern and Synaptic Circuitry of Cholinergic Neurons in the Rat Retina / 대한해부학회지

The role of acetylcholine as an excitatory neurotransmitter is well established, and cholinergic neurons appear to play an important role in the mammalian retinae. Though it has been reported that certain conventional and displaced amacrine cells are consistently labeled with anti-choline acetyltransferase antiserum in the mammalian retinae, little has been studied on the synaptic circuitry of cholinergic neurons to clarify mechanism of its action in the visual processing of the mammalian retinae. This study was conducted to localize cholinergic neurons and to define their synaptic circuitry in the rat retina by immunocytochemical method using anti-choline acetyltransferase antiserum. The results were as follows: 1. Cholinergic neurons of the rat retina were conventional amacrine cells located in the inner nuclear layer and displaced amacrine cells in the ganglion cell layer. 2. Cholinergic amacrine cells were branched in the middle of the sublamina a of the inner plexiform layer, and cholinergic displaced amacrine cells branched in the sublamina b, forming one prominent band, respectively. 3. Presynaptic processes to cholinergic amacrine cell processes were axon terminals of invaginating and flat cone bipolar cells, and unlabelled amacrine cell processes in the inner plexiform layer. Postsynaptic dyads at the ribbon synapses of axon terminals of cone bipolar cells were cholinergic amacrine cell process and dendrite of ganglion cell, cholinergic amacrine cell process and unlabelled amacrine cell process and cholinergic amacrine cell process and cholinergic amacrine cell process. In addition, cholinergic amacrine cell process formed postsynaptic monoad at the ribbon synapse. 4. Cholinergic amacrine cell processes made output conventional chemical synapses onto the dendrites of ganglion cells, unlabelled amacrine cell processes and cholinergic amacrine cell processes in the inner plexiform layer. These results demonstrate that (1) cholinergic neurons are conventional amacrine cells and displaced amacrine cells of which somata are located in the inner nuclear layer and ganglion cell layer, respectively, (2) cholinergic conventional amacrine cells are involved in OFF pathway, and cholinergic displaced amacrine cells play an important role in ON pathway in visual processing of lightness, and (3) acetylcholine released from cholinergic neurons by light excites directly ON and OFF ganglion cells or indirectly ON and OFF ganglion cells via non-cholinergic amacrine cells.

Synaptic Diversity in the Neostriatum of the Cat / 대한해부학회지

An attempt has been made to discriminate synaptic diversity in the neostriatum of the cat with emphasis on the characteristic structures of axon terminals and postsynaptic profiles. The differentiation of the axon terminals was based on the size and shape of synaptic vesicles in the axoplasm. Three types of axon terminals could be differentiated: Type I, the terminals contained small round (45 nm in diameter) vesicles; type II, the terminals with large pleomorphic (50 nm) vesicles; and type III, the terminals contained flattened (45 x 25 nm) vesicles. The type I terminals were making asymmetrical or symmetrical synapses in contact with the somata, dendrites and dendritic spines of neurons in the neostriatum, and a few type I terminals making asymmetrical or symmetrical contact with axons were also observed. The type II and III terminals were making symmetrical contact with the somata and dendrites of neostriatal neurons. A few type II terminals formed at the node of Ranvier of myelinated nerve fibers were making symmetrical contact with large dendrites. Additionally, dendro-dendritic and serial syanpses were rarely found in the neostriatum. In the serial synapses composed of axo-dendritic and dendro-dendritic synapses, the type I terminals making asymmetrical contact and the type II making symmetrical contact were identified.

Topographic distribution, ultrastructure and synaptic organization of dopaminergic neurons in the retina of redents II. ultrastructure and synaptic organization / 대한해부학회지

No abstract available.

Double label immunocytochemistry for dopaminergic and parvalbuminergic neurons using diaminobenzidine and benzidine dihydrochloride in the rat substantia nigra / 대한해부학회지

No abstract available.

Totpgraphic distribution. ultrastructure and synaptic organization of dopaminergic neurons in the retinae of rodents: I. topographic distribution / 대한해부학회지

No abstract available.