Retrogression of the metabolic activity of astrocytes after extinction of an experimental epileptogenic focus. Histochemical investigation

Previous anatomochemical and experimental investigations had resulted in the histochemical characterisation of the epileptogenic focus by the presence of "activated astrocytes"; cortical reactive astrocytes acquiring an intense activity of sundry dehydrogenases (DH) and becoming visible in spite of the positivity of the neuropil, and stopping at the amylose step in the synthesis of glycogen. This activation exists from the first electrical sings of epilepsy and even preceeds them in the case of glucose-6-phosphate DH. The present study deals with the fate of activated astrocytes after the disappearance of electrical signs of the epileptogenic focus produced by implantation of a cobalt powder-gelatin pellet into the cerebral cortex of the rat. After extinction of the focus, high activity of DH persists in astrocytes only in the immediate vicinity of the glial scar surrounding the implant. Later, among the DH investigated only glutamate and specially glucose-6-phosphate DH maintain an activity intense enough to reveal these cells on the background of the neuropil. These results point to a progressive return to a normal metabolism of astrocytes after the disappearance of the electrical signs of epilepsy by way of steps similar to those preceeding these signs.     

小鼠大脑皮质星形胶质细胞原代培养与鉴定

为探讨简便、高效的大脑皮质星形胶质细胞体外培养方法,本研究取新生24 h内的ICR小鼠大脑皮层,采用物理方法将其分成约1 mm^3,震荡过滤后进行培养。通过拍照的方式记录原代培养1 d、3 d、7 d、14 d、21 d、28 d、35 d和原代培养14 d后再传代培养14 d(记为P2-14 d)细胞形态;通过实时定量PCR和Western blotting比较原代培养1周、2周、3周、4周、5周和原代培养2周后再传代培养2周(即P2-2)的星形胶质细胞内胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)基因和蛋白水平变化。选取GFAP、S100-β和谷氨酸转运蛋白(excitatory amino acid transporter 1,EAAT1)标记星形胶质细胞,微管相关蛋白(microtubuleassociated protein 2,MAP-2)、离子钙接头蛋白-1(ionized calcium-binding adapter molecule 1,Iba-1)和髓鞘相关糖蛋白(myelin associated glycoprotein,MAG)抗体分别标记神经元、小胶质细胞和少突胶质细胞。通过免疫荧光染色鉴定细胞种类及纯度。研究结果显示细胞生长良好,原代培养4周星形胶质细胞内GFAP比2周、3周、5周和传代培养2周的细胞更加稳定。经免疫荧光鉴定,星形胶质细胞纯度在95%以上。本实验采用相对较简单经济的方法培养出高纯度且生理状态相对较稳定的原代星形胶质细胞,该细胞模型不仅可以用于星形胶质细胞生理功能研究,还可以用于中枢神经系统相关疾病的体外研究。     

FINE STRUCTURE OF THE SURFACE OF THE CEREBRAL CORTEX OF HUMAN BRAIN

Evidence is presented for the existence of arborizing cytoplasmic processes extending from the surface of the cerebral cortex of human brain into the surrounding fluid medium. These originate from subpial fibrous astrocytes and contain the usual cytoplasmic organelles of those cells. They are bordered by basement membrane. Their occurrence is localized and variable over the cortical surface. They are more prevalent in pathological human material than in "normal" human brain and somewhat more prevalent in the latter than in normal rat cortex. Some additional information is presented regarding the relationship of leptomeninges to the cortical surface. The pia mater does not invariably adhere inseparably to the subjacent layer of fibrous astrocytes as generally assumed at present, nor does it always form a continuous layer over the surface of the brain in the material under study. Both collagen and cytoplasmic extensions of astrocytes intervene between these layers. These findings imply that glial elements of the cortex have direct access to the cerebrospinal fluid.     

Regional difference of glutamate-induced swelling in cultured rat brain astrocytes

L-glutamate (glutamate) is an important neurotoxin as well as the major excitatory neurotransmitter. Extracellular glutamate levels are elevated following ischemia, hypoglycemia, and trauma. One consequence of elevated glutamate levels is cell swelling. Such swelling occurs primarily in astroglial cells. We characterized the regional difference in glutamate-induced swelling response of cultured astrocytes from rat cerebral cortex, hippocampus and cerebellum. Glutamate produced dose-dependent astrocytic swelling in both cerebral cortex and hippocampus, showing a maximal effect in 0.5 mM concentration, as measured by 3-O-methyl-D-[1-3H]glucose uptake. However, in cerebellum, glutamate did not produce astrocytic swelling. It has been suggested that Na+ -dependent glutamate uptake is a possible mechanism of glutamate-induced swelling. The Vmax for glutamate uptake into cerebellum astrocytes was significantly lower (6.7 nmol/mg protein/min) than those for cerebral cortex and hippocampus astrocytes (13.0 and 12.0 nmol/mg protein/min, respectively). In three regions, more than 90% of the cultured cells showed glial fibrillary acidic protein (GFAP) immunoreactivity. Immunoreactivity of GLT, one of the markers of glutamate transporters, which is expressed at low levels in cultured astrocytes, did not show any differences in three regions. However, immunoreactivities of GLAST, the other astroglial glutamate transporter, and aquaporin4 (APQ4), a water transporter, were significantly higher in cerebral cortex and hippocampus than in cerebellum. These results may explain the regional difference of glutamate-induced astrocytic swelling.     

Expression of thrombospondin-1 and CD36 and CD47 receptors in the rat brain after exposure to damaging factors in the early postnatal period

The expression of thrombospondin-1 (TS-1) and its receptors CD47 and CD36 in the cerebral cortex and hippocampus of rats under damaging factors in the early postnatal period was studied. After hypoxia on the 7th day of postnatal development, an increase in the number of CD47-expressing cerebral endothelial cells (days of postnatal development: P28–P70) and reduction in the number of TS-1-expressing astrocytes in the cortex at P28 were observed. In animals subjected to early postnatal stress at the age of P2–P15, a decrease in TS-1-expressing astrocytes in the cortex and hippocampus was registered (predominantly at the age of P28). It was noted that these changes characterize the period of long-term effects (P28–P70) of early stress that is relevant to the processes of reparative angiogenesis and arresting of neurological deficits.     

Glial Fibrillary Acidic Protein is Greatly Modified by Oxidative Stress in Aceruloplasminemia Brain

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism caused by mutations in the ceruloplasmin (Cp) gene. The neuropathological hallmark of this disease is intracellular iron overload, which is thought to lead to neuronal cell death through increased oxidative stress. We evaluated and characterized protein oxidation in the brain of a patient with this disease. The protein carbonyl content in the cerebral cortex of the patient was elevated compared to controls. Furthermore, peptide mass fingerprinting and partial amino acid sequencing identified glial fibrillary acidic protein (GFAP) as the major carbonylated protein in the cerebral cortex of the patient. In conjunction with the facts that Cp mainly localizes to astrocytes in the central nervous system and that astrocytes are loaded with much more iron than neurons in the cerebral cortex, our findings indicate that Cp deficiency may primarily damage astrocytes. We speculate that the dysfunction of astrocytes may be causatively related to neuronal cell loss in aceruloplasminemia.     

Glial fibrillary acidic protein is greatly modified by oxidative stress in aceruloplasminemia brain

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism caused by mutations in the ceruloplasmin (Cp) gene. The neuropathological hallmark of this disease is intracellular iron overload, which is thought to lead to neuronal cell death through increased oxidative stress. We evaluated and characterized protein oxidation in the brain of a patient with this disease. The protein carbonyl content in the cerebral cortex of the patient was elevated compared to controls. Furthermore, peptide mass fingerprinting and partial amino acid sequencing identified glial fibrillary acidic protein (GFAP) as the major carbonylated protein in the cerebral cortex of the patient. In conjunction with the facts that Cp mainly localizes to astrocytes in the central nervous system and that astrocytes are loaded with much more iron than neurons in the cerebral cortex, our findings indicate that Cp deficiency may primarily damage astrocytes. We speculate that the dysfunction of astrocytes may be causatively related to neuronal cell loss in aceruloplasminemia.     

Differential expression of isoforms of the regulatory subunit of type II cAMP-dependent protein kinase in rat neurons, astrocytes, and oligodendrocytes

The RII-B isoform of the regulatory subunit (R) of cAMP-dependent protein kinase II is abundantly and selectively expressed in cerebral cortex (Erlichman, J., Sarkar, D., Fleischer, N., and Rubin, C. S. (1980) J. Biol. Chem. 255, 8179-8184). In contrast to the cytosolic RII-H isoform from heart and other non-neural tissues, a substantial fraction of cerebral cortex RII-B is tightly associated with cell organelles. In order to study the cellular basis for the localization and abundance of RII-B in this complex and heterogeneous tissue, rat cerebral cortex was fractionated into highly purified populations of neurons, astrocytes, and oligodendrocytes. In neurons and astrocytes more than 80% of the total cAMP-binding activity is contributed by RII subunits, whereas the myelin-producing oligodendrocytes contain nearly equal proportions of RI (from protein kinase I) and RII. Approximately 70% of RII and RI subunits are associated with the particulate fraction in each of the three types of brain cells. The nature of the RII isoforms expressed in the cytosolic and particulate fractions of the purified brain cells was established by performing Western immunoblot and indirect immunoprecipitation analyses with selective and sensitive polyclonal antibodies directed against RII-B. Astrocytes and neurons exhibit high levels of RII-B, whereas oligodendrocytes contain the RII-H isoform. Thus, the expression of RII isoforms is not uniform among brain cells that are anatomically and developmentally related. Rather, it appears that RII-B and RII-H are expressed in a cell-specific fashion within cerebral cortex and this might reflect an RII-mediated adaptation of protein kinase II to the specialized metabolic and functional roles of neurons, astrocytes, and oligodendrocytes.     

Development of the various glial cell types in the cerebral cortex of postnatal rats

The present quantitative study in the postnatal rats showed the rapid growth of the various glial cell types in the cerebral cortex. Among them, the increase of microglia was most dramatic. The increase was about 15 times, covering a period of 15 days extending from 5 days of age to 20 days. The majority of the microglia observed were in the outer third of the cortex. During the same period, the number of oligodendrocytes and astrocytes also showed a steady but moderate increase. The increase of oligodendrocytes was most significant between 5 and 10 days. Their density was greater in the inner third of the cortex. Astrocytes were distributed uniformly throughout. Examination of the cerebral cortex in 1- to 3-day-old rats by electron microscopy showed sporadic ameboid microglia cells and glioblasts. The possibility that they served as the precursor cells of microglia and macroglia (astrocytes and oligodendrocytes), respectively, was considered.     

Metabolism and Release of Glutamate in Cerebellar Granule Cells Cocultured with Astrocytes from Cerebellum or Cerebral Cortex

Cerebellar granule cells were cocultured with astrocytes from either cerebral cortex or cerebellum in two different systems. In one system the cells were plated next to each other only sharing the culture medium (separated cocultures) and in the other system the granule cells were plated on top of a preformed layer of astrocytes (sandwich cocultures). Using astrocytes from cerebellum, granule cells developed morphologically and functionally showing a characteristic high activity of the glutamate synthesizing enzyme aspartate aminotransferase (AAT) as well as a high stimulus-coupled transmitter release regardless of the culture system, i.e., granule cells could grow on top of cerebellar astrocytes as well as next to these cells. In the case of cerebral cortex astrocytes it was found that cerebellar granule cells did not develop (11% survival) when seeded on top of these astrocytes. This was indicated by the morphological appearance of the cultures as well as by a negligible difference between the AAT activity in sandwich cocultures and astrocytes cultured alone. On the other hand, granule cells in separated cocultures with cerebral cortex astrocytes exhibited a normal morphology and a high activity of AAT as well as a large stimulus-coupled transmitter release. Cerebellar and cortical astrocytes expressed the astrocyte specific enzyme glutamine synthetase in a glucocorticoid-inducible form regardless of the culture system. The results show that under conditions of direct contact between granule cells and astrocytes, regional specificity exists with regard to neuron-glia contacts. This specificity does not seem to involve soluble factors present in the culture medium because in separated cocultures the cerebellar granule cells developed normally regardless of the regional origin of the astrocytes.     

The regulation of proenkephalin expression in a distinct population of glial cells.

The expression of opioid genes was examined in isolated populations of glial cells in primary culture. Northern blot analysis of purified type I astrocytes, oligodendrocytes and mixed oligodendrocyte-type-2-astrocyte lineage cells derived from cerebral cortex demonstrated robust expression of proenkephalin mRNA exclusively in type I astrocytes. The expression of proenkephalin mRNA was stimulated by the beta-adrenergic agonist isoproterenol, and 8-(4-chlorophenyl thio)adenosine 3'-5'-cyclic monophosphate (cpt-cAMP). Both of these compounds regulated a proenkephalin-chloramphenicol acetyltransferase fusion gene transiently transfected into type I astrocytes. HPLC and immunoassay of the cell culture media revealed significant levels of unprocessed proenkephalin secreted by the cell and this secretion was stimulated by isoproterenol and cpt-cAMP. The relatively high levels of proenkephalin expressed suggest that enhanced expression in astrocytes may be important during neural development, in trauma-induced gliosis and in neuroimmune interactions.     

Producing the cortical phenomenon of "kindling" during acute experiments on cats

Acute experiments on cats enabled spontaneous epileptogenic foci to be produced by means of electrical stimulation of the cerebral cortex. The central area of the suprasylvian gyrus was stimulated by 5-sec stimuli at 3-sec intervals. The strength of neuronal response gradually increased until spontaneous electrical activity set in as stimulation was set at a fixed intensity which had originally evoked local residual discharge at stimulation foci only, i.e., the phenomenon of "kindling" was observed. When "kindling" was produced in the cortical area under study, bursts of spindle activity were recorded on all ECoG with increasing frequency. Recordings of spindle activity then changed to a "spike-wave" pattern of activity. The results of these investigations, which were performed on an isolated strip of cortex, point to the involvement of subcortical structures in mediating "kindling" of the cortical focus.Institute of Clinical and Experimental Neurology, Ministry of Public Health of the Georgian SSR, Tbilisi. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 601–606, September–October, 1985.     

Truncated tyrosine kinase B brain-derived neurotrophic factor receptor directs cortical neural stem cells to a glial cell fate by a novel signaling mechanism

During development of the mammalian cerebral cortex neural stem cells (NSC) first generate neurons and subsequently produce glial cells. The mechanism(s) responsible for this developmental shift from neurogenesis to gliogenesis is unknown. Brain-derived neurotrophic factor (BDNF) is believed to play important roles in the development of the mammalian cerebral cortex; it enhances neurogenesis and promotes the differentiation and survival of newly generated neurons. Here, we provide evidence that a truncated form of the BDNF receptor tyrosine kinase B (trkB-t) plays a pivotal role in directing embryonic mouse cortical NSC to a glial cell fate. Expression of trkB-t promotes differentiation of NSC toward astrocytes while inhibiting neurogenesis both in cell culture and in vivo. The mechanism by which trkB-t induces astrocyte genesis is not simply the result of inhibition of full-length receptor with intrinsic tyrosine kinase activity signaling. Instead, binding of BDNF to trkB-t activates a signaling pathway (involving a G-protein and protein kinase C) that induced NSC to become glial progenitors and astrocytes. Thus, the increased expression of trkB-t in the embryonic cerebral cortex that occurs coincident with astrocyte production plays a pivotal role in the developmental transition from neurogenesis to gliogenesis. Our findings suggest a mechanism by which a single factor (BDNF) regulates the production of the two major cell types in the mammalian cerebral cortex.     

Glucagon-like peptide-2 stimulates the proliferation of cultured rat astrocytes.

Glucagon-like peptide-2 (GLP-2) is a potent intestinotrophic/satiety hormone that acts through a G protein-coupled receptor. To determine whether or not GLP-2 has any effect on cellular proliferation on neural cells, we examined the effects of this peptide on cultured astrocytes from rat cerebral cortex. The expression of the GLP-2 receptor gene in both cerebral cortex and astrocytes was determined by RT-PCR and Southern blotting. Also, cells responded to GLP-2, producing cAMP in a dose-dependent manner (EC50 = 0.86 nm). GLP-2 also stimulated the DNA synthesis rate in rat astrocytes. When proliferation was assessed by measuring [3H]thymidine incorporation into DNA or staining cells with crystal violet, GLP-2 produced a dose-dependent increase in both parameters. Similarly, when the numbers of cells in different phases of the cell cycle were measured by flow cytometry, a dose-dependent decrease in those in the G0-G1 phase and an increase in those in the S and G2-M phases were observed after 24 h incubation with GLP-2. By contrast, the number of hypodiploid cells was not affected during the experimental time. Also, GLP-2 produced a significant increase in the mRNAs of c-fos and c-jun when gene expression was determined by Northern blotting. These results suggest that GLP-2 directly stimulates the proliferation of rat astrocytes; this may open new insights in the physiological role of this novel neuropeptide.     

Cerebral Blood Flow Modulation by Basal Forebrain or Whisker Stimulation Can Occur Independently of Large Cytosolic Ca2+ Signaling in Astrocytes

We report that a brief electrical stimulation of the nucleus basalis of Meynert (NBM), the primary source of cholinergic projection to the cerebral cortex, induces a biphasic cerebral cortical blood flow (CBF) response in the somatosensory cortex of C57BL/6J mice. This CBF response, measured by laser Doppler flowmetry, was attenuated by the muscarinic type acetylcholine receptor antagonist atropine, suggesting a possible involvement of astrocytes in this type of CBF modulation. However, we find that IP3R2 knockout mice, which lack cytosolic Ca2+ surges in astrocytes, show similar CBF changes. Moreover, whisker stimulation resulted in similar degrees of CBF increase in IP3R2 knockout mice and the background strain C57BL/6J. Our results show that neural activity-driven CBF modulation could occur without large cytosolic increases of Ca2+ in astrocytes.     

Neuroactive Amino Acids in Focally Epileptic Human Brain: A Review

Studies of neuroactive amino acids and their regulatory enzymes in surgically excised focally epileptic human brain are reviewed. Concentrations of glutamate, aspartate and glycine are significantly increased in epileptogenic cerebral cortex. The activities of the enzymes, glutamate dehydrogenase and aspartate aminotransferase, involved in glutamate and aspartate metabolism are also increased. Polyamine synthesis is enhanced in epileptogenic cortex and may contribute to the activation of N-methyl-D-aspartate (NMDA) receptors. Nuclear magnetic resonance spectroscopy (NMRS) reveals that patients with poorly controlled complex partial seizures have a significant diminution in occipital lobe gamma aminobutyric acid (GABA) concentration. The activity of the enzyme GABA-aminotransaminase (GABA-T) which catalyzes GABA degredation is not altered in epileptogenic cortex. NMRS studies show that vigabatrin, a GABA-T inhibitor and effective antiepileptic, significantly increases brain GABA. Glutamate decarboxylase (GAD), responsible for GABA synthesis, is diminished in interneurons in discrete regions of epileptogenic cortex and hippocampus. In vivo microdialysis performed in epilepsy surgery patients provides measurements of extracellular amino acid levels during spontaneous seizures. Glutamate concentrations are higher in epileptic hippocampi and increase before seizure onset reaching potentially excitotoxic levels. Frontal or temporal cortical epileptogenic foci also release aspartate, glutamate and serine particularly during intense seizures or status epilepticus. GABA in contrast, exhibits a delayed and feeble rise in the epileptic hippocampus possibly due to a reduction in the number and/or efficiency of GABA transporters.     

Low temperature induced de-differentiation of astrocytes

Radial glial cells are astrocyte precursors, which are transiently present in the developing central nervous system and transform eventually into astrocytes in the cerebral cortex and into Bergmann glia in the cerebellum. Previous reports indicate that the transformation from radial glia to astrocytes can be reversed by diffusible chemical signals derived from embryonic forebrain in vitro and by freezing injury in vivo. But there is no direct evidence proving that mature astrocytes can de-differentiate into radial glial cells. Here we show that purified astrocytes could de-differentiate into radial glial-like cells (RGLCs) in vitro with freeze-thaw stimulation. RGLCs had the expression of markers for radial glia including Nestin and Pax6, and astrocyte markers, the glial fibrillary acidic protein and Vimentin. Cortical neurons, when co-cultured with RGLCs, migrated along the processes of RGLCs at an average speed of 26.26 +/- 3.36 microm/h. Moreover, the proliferation of RGLCs was significantly promoted by epidermal growth factor (EGF) at the concentration of 10-30 ng/ml. These results reveal that low temperature induces astrocytes to de-differentiate into immature RGLCs, which provides an in vitro model to investigate mechanisms of astroglial cells de-differentiation.     

Expression and potential role of phospholipase D1 in cryoinjured cerebral cortex of rats

The expression and potential role of phospholipase D1 (PLD1) were studied in the cerebral cortex of rats after freeze injury. Histopathologically, cryoinjury, by exposing cerebral cortex to a prechilled rod for 1 minute, produced consistent pathological lesions, specifically neuronal death, infiltration of macrophages into the center of the cryoinjury, and reactive astrogliosis at the periphery, which caused the lesion site to become encased. Western blot analysis showed that PLD1 expression in the ipsilateral cerebral cortex increased significantly during days 1 to 3 after cryoinjury and declined slightly at post-injury day 7. PLD1 immunoreactivity was very low in the brains of sham-operated control adults. After cryoinjury, there was substantial PLD1 immunostaining of numerous inflammatory cells in the ipsilateral cortex, which were identical to ED1-positive macrophages. In addition, PLD1 immunoreactivity was increased in some neurons and astrocytes at the periphery of the cryoinjury at post-injury days 3 and 7. These findings suggest that cryoinjury by means of prechilled rods induced consistent histopathological changes in the cerebral cortex. In addition, expression of a cell activation signal, PLD1, was upregulated in macrophages and astrocytes in the ipsilateral cerebral cortex after cryoinjury.     

Cultured Rat Astrocytes Give Rise to Neural Stem Cells

Previously, we reported the occurrence of neural stem cells (NSCs) around an area of damage after rat traumatic brain injury (TBI), but it was unclear if this was due to blastgenesis in astrocytes, or to NSCs migrating from the subventricular zone (SVZ). In this study, NSCs were isolated and cultured from cultured type 1 astrocytes taken from newborn rat cortex in which the subventricular zone and hippocampus had been discarded. All cultured type 1 astrocytes showed glial fibrillary acidic protein (GFAP) immunopositivity. Nestin immunopositive spheres were isolated from type 1 astrocytes and cultured in the presence of bFGF and EGF in the medium. Neurospheres differentiated into Tuj1-, GFAP- and A2B5-positive cells after 4 days of culture without bFGF and EGF. These results indicate that isolated neurospheres from brain cortex astrocytes can differentiate into neurons and glia and might contribute to neurogenesis and neuroplasticity.