Spatial arrangement of microglia in the mouse hippocampus: a stereological study in comparison with astrocytes

Microglia are classically considered to be immune cells in the brain, but have now been proven to be involved in neuronal activity as well. Here we stereologically analyzed the spatial arrangement of microglia in the mouse hippocampus. First, we estimated the numerical densities (NDs) of microglia identified by ionized calcium-binding adaptor molecule 1 (Iba1). Despite that microglia appeared to be evenly distributed throughout the hippocampal area, the NDs demonstrated significant dorsoventral, interregional, and interlaminar differences. Briefly, the NDs in the ventral hippocampus were significantly lower in the CA3 region than in the CA1 region and dentate gyrus, although no interregional differences were detectable in the dorsal hippocampus. Both in the CA1 and CA3 regions, the NDs were significantly higher in the stratum lacunosum-moleculare than in the remaining layers. Next, we investigated the spatial patterns of distribution of Iba1-labeled microglia and S100beta-labeled astrocytes. So far as we examined, the somato-somatic contacts were not seen among microglia or among astrocytes, whereas the close apposition between microglia and astrocytes were occasionally detected. The 3D point process analysis showed that the spatial distribution of microglia was significantly repulsive. Because the statistical territory of single microglia was larger than that estimated from process tracing, they are not likely to touch each other with their processes. Astrocytes were distributed slightly repulsively with overlapping areas. The 3D point process analysis also revealed a significant spatial attraction between microglia and astrocytes. The present findings provide a novel anatomical basis for glial research.     

Distribution of P2X receptors on astrocytes in juvenile rat hippocampus

Recent evidence suggested that ATP acting via ionotropic (P2X) and metabotropic (P2Y) purinergic receptors might be involved in signaling between glial cells and within glial-neuronal networks. In contrast to their neuronal counterpart, the identity of P2X receptors in CNS glial cells is largely unknown. In the present study, antibodies recognizing the subunits P2X1-P2X7 were applied together with the astroglial marker S100beta and nuclear labeling with Hoechst 33342 to investigate semiquantitatively the distribution of the whole set of P2X receptors in astrocytes of the juvenile rat hippocampus. Expression of P2X1-P2X4, P2X6, and P2X7 subunits was observed in astrocytes of various hippocampal subregions, but the cells were completely devoid of P2X5 protein. S100beta-positive cells expressing subunits P2X3-P2X7 occurred evenly in the different subfields, while P2X1- and P2X2-positive astrocytes were distributed more heterogeneously. The staining pattern of P2X subunits also differed at the subcellular level. Antibodies against P2X2 and P2X4 labeled both astroglial cell bodies and processes. Immunoreactivity for P2X1 and P2X6 was mainly confined to somatic areas of S100beta-positive cells, whereas the subunit P2X3 was primarily localized along astroglial processes. Knowledge of the distribution of P2X receptors might provide a basis for a better understanding of their specific role in cell-cell signaling.     

Reaction of mouse brain oligodendrocytes and their precursors, astrocytes and microglia, to proinflammatory mediators circulating in the cerebrospinal fluid

The response of glial cells to the acute intracerebroventricular administration of interferon-gamma, and of this cytokine combined with the endotoxin lipopolysaccharide or with tumor necrosis factor-alpha, was investigated in the brain of adult mice over a time course of 1 week. Oligodendrocytes were identified by immunocytochemistry, using O4 to label their precursors and 2',3'-cyclic nucleotide 3'-phosphohydrolase as marker of mature cells. Astrocytes were labeled by glial fibrillary acidic protein immunoreactivity and microglial cells by tomato lectin histochemistry. Compared with ovalbumin-injected control cases, all cytokine treatments caused a marked decrease of immunostained mature oligodendrocytes in the brain since 1 day postinjection. O4+ oligodendrocyte precursors increased instead progressively from 2 to 7 days. Astrocytes, markedly activated by cytokine treatments, also exhibited a progressive quantitative increase from 2 days onward. Activation and proliferation of microglial cells were instead most evident at 24 h postinjection. Such glial responses to interferon-gamma injections were especially marked in the periventricular brain parenchyma and were enhanced by coadministration of lipopolysaccharide or tumor necrosis factor-alpha. The findings show that a pulse of proinflammatory mediators in the cerebrospinal fluid affects mature oligodendrocytes, concomitantly with the early appearance of activated microglia, and that such reactions are rapidly followed by an increase of oligodendrocyte precursors paralleled by astrocytic activation. The data, which allowed dissecting the events elicited in glial cell populations by inflammatory mediators via the cerebrospinal fluid, indicate that these molecules elicit in vivo a toxic effect on mature oligodendrocytes and a stimulation of their precursors in the adult brain.     

Neurons modulate oxytocin receptor expression in rat cultured astrocytes: involvement of TGF-beta and membrane components.

We examined the effect of neurons on oxytocin (OT) receptors (OTR) and OTR gene expression in cultured astrocytes. The addition of neuron-conditioned medium induced an increase of both OTR binding and OTR mRNA level. This effect was enhanced after the medium was boiled or acidified. As it is known that transforming growth factor-beta (TGF-beta) can be released from carrier proteins by acid or heat, TGF-beta1 and 2 were tested and found to induce an increase of OTR binding. Furthermore, TGF-beta antibody abolished the stimulatory effect of normal or acidified neuron-conditioned medium. Neurons added to cultured astrocytes without contact mimicked the stimulatory effect of the conditioned medium. In contrast, neurons added with contact, induced a decrease in OTR binding and an increase of mRNA level, whereas neuronal membranes induced a decrease of both OTR binding and mRNA levels. In conclusion, the present data demonstrate that in vitro, neurons are able to modulate astrocytic OTR expression at the level of both protein and mRNA. They stimulate this expression through their release of TGF-beta and inhibit it by the action of unknown membrane components.     

Astrocytes contribute to neuronal impairment in beta A toxicity increasing apoptosis in rat hippocampal neurons

Astrocytosis is a common feature of amyloid plaques, the hallmark of Alzheimer's disease (AD), along with activated microglia, neurofibrillary tangles, and beta-amyloid (beta A) deposition. However, the relationship between astrocytosis and neurodegeneration remains unclear. To assess whether beta A-stimulated astrocytes can damage neurons and contribute to beta A neurotoxicity, we studied the effects of beta A treatment in astrocytic/neuronal co-cultures, obtained from rat embryonic brain tissue. We found that in neuronal cultures conditioned by beta A-treated astrocytes, but not directly in contact with beta A, the number of apoptotic cells increased, doubling the values of controls. In astrocytes, beta A did not cause astrocytic cell death, nor did produce changes in nitric oxide or prostaglandin E(2) levels. In contrast, S-100 beta expression was remarkably increased. Our data show for the first time that beta A--astrocytic interaction produces a detrimental effect on neurons, which may contribute to neurodegeneration in AD.     

S100b counteracts effects of the neurotoxicant trimethyltin on astrocytes and microglia

Central nervous system degenerative diseases are often characterized by an early, strong reaction of astrocytes and microglia. Both these cell types can play a double role, protecting neurons against degeneration through the synthesis and secretion of trophic factors or inducing degeneration through the secretion of toxic molecules. Therefore, we studied the effects of S100B and trimethyltin (TMT) on human astrocytes and microglia with two glial models, primary cultures of human fetal astrocytes and a microglia cell line. After treatment with 10(-5) M TMT, astrocytes showed morphological alterations associated with an increase in glial fibrillary acidic protein (GFAP) expression and changes in GFAP filament organization. Administration of S100B before TMT treatment prevented TMT-induced changes in morphology and GFAP expression. A decrease in inducible nitric oxide synthase expression was observed in astrocytes treated with TMT, whereas the same treatment induced iNOS expression in microglia. In both cases, S100B prevented TMT-induced changes. Tumor necrosis factor-alpha mRNA expression in astrocytes was not modified by TMT treatment, whereas it was increased in microglia cells. S100B pretreatment blocked the TMT-induced increase in TNF-alpha expression in microglia. To trace the mechanisms involved in S100B activity, the effect of BAY 11-7082, an inhibitor of nuclear factor-kappaB (NF-kappaB) activation, and of PD98059, an inhibitor of MEK-ERK1/2, were investigated. Results showed that the protective effects of S100B against TMT toxicity in astrocytes depend on NF-kappaB, but not on ERK1/2 activation. These results might help in understanding the role played by glial cells in brain injury after exposure to chemical neurotoxicants and support the view that S100B may protect brain cells in case of injury. (c) 2005 Wiley-Liss, Inc.     

Binding of an antibody against a noncompact myelin protein to presumptive glial cells in the visual system of the crab Ucides cordatus

Glial cells, in both vertebrate and invertebrate nervous systems, provide an essential environment for developmental, supportive, and physiological functions. However, information on glial cells themselves and on glial cell markers, with the exception of those of Drosophila and other insects, is not abundant in invertebrate organisms. A common ultrastructural feature of invertebrate nervous systems is that layers of glial cell cytoplasm-rich processes ensheath axons and neuronal and glial somata. In the present study, we have examined the binding of a monoclonal antibody to 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in the compound eye and optic lobe of the crab Ucides cordatus using both light and electron microscopy. CNPase is a noncompact myelin protein that is a phenotypic marker of oligodendroglial and Schwann cells, is apparently involved in the ensheathment step prior to myelin compaction, and is also expressed by the potentially myelinating olfactory ensheathing glia. CNPase has raised much interest, first by virtue of its unusual enzymatic activity and more recently by its membrane-skeletal features and possible involvement in migration or expansion of membranes. We have found CNPase-like immunoreactivity in most cells of the compound eye basement membrane and both in optic cartridges of the synaptic layer and cells of the outer sublayer of the lamina ganglionaris. The results suggest that in the crab visual system some, but not all, glial cells, including some adaxonal glia, may express the noncompact myelin protein CNPase or a related protein.     

Alpha 1 and alpha 2 Adrenergic receptors in mouse brain astrocytes from primary cultures

Mouse brain astrocytes from primary cultures were found to contain both alpha 1 and alpha 2 adrenergic receptors. 3H WB 4101 labeled one category of binding site (KD = 1.5 +/- 0.39 nM, Bmax = 64 +/- 7.9 fmoles/mg protein) with typical alpha 1 adrenergic specificity (WB 4101 greater than prazosin greater than yohimbine). The density of alpha 1 adrenergic receptors was 2-3 times higher in mouse cerebral cortex than in glial cells. Like rat brain [U'Pritchard et al, 1979; Rouot et al, 1980], mouse glial cells were found to contain two categories of 3H clonidine binding sites: high affinity sites, which were identical to the high but not to the low affinity sites found in rat brain, since 1) they displayed the same affinity for 3H clonidine (KD = 1.2 +/- 0.13 nM, n = 4) and the same typical alpha 2 adrenergic specificity (yohimbine greater than WB 4101 greater than prazosin); 2) the dissociation rate constant for clonidine binding was equal to 0.06 min-1, a value close to that found previously for the high affinity 3H clonidine binding sites in rat brain (0.05 min-1); and 3) divalent cations augmented and guanyl nucleotides reduced 3H clonidine binding as in rat brain. Na+ decreased 3H clonidine binding in a complex manner. The number of high affinity sites in glial cells (52 +/- 9.4 fmoles/mg protein, n = 4) was half the number found in mouse cerebral cortex (98 fmoles/mg protein). Low affinity 3H clonidine binding sites (KD = 81 +/- 18 nM, Bmax = 96 +/- 5.8 fmoles/mg protein, n = 3) were not fully characterized. In conclusion, glial cells contained the same alpha adrenergic receptors as those described in brain, but their physiological function is not yet known.     

Rapid up-regulation of the neuronal serotoninergic phenotype by brain-derived neurotrophic factor and cyclic adenosine monophosphate: relations with raphe astrocytes

Up-regulation of the neuronal serotoninergic phenotype in relation to astrocytic population was studied in primary cultures of rat embryonic rostral raphe. Short treatments (18 hr at day in vitro 4) with brain-derived neurotrophic factor (BDNF) or dibutyryl-cAMP (dBcAMP) increased the number of serotoninergic neurons by approximately 80% and approximately 40%, respectively, and markedly enhanced the branching (by 11-fold and 5-fold, respectively) and total length (by 4-fold and 2.5-fold, respectively) of their neurites. Concomitantly, under BDNF treatment, the astrocyte population was decreased by half and became mostly protoplasmic-like. In contrast, dBcAMP treatment also reduced the astrocytic cell density (by one-third) but induced a stellate morphology. Similar short treatment with the astrocyte-derived S100beta factor induced no modification of the serotonin (5-HT) neuronal phenotype nor of astrocytes morphology. Both BDNF- and cAMP-induced effects were abolished by simultaneous treatment with the specific tyrosine kinase inhibitor genistein, suggesting a role for the high-affinity BDNF receptor tyrosine kinase (TrkB). These data suggest that BDNF and cAMP, but not S100beta, rapidly induce both an up-regulation of the 5-HT neuronal phenotype and modifications of the neighboring astrocytes in a TrkB-dependent manner.     

Muscarinic receptor M(2) in cat visual cortex: laminar distribution, relationship to gamma-aminobutyric acidergic neurons, and effect of cingulate lesions.

Acetylcholine can have diverse effects on visual cortical neurons as a result of variations in postsynaptic receptor subtypes as well as the types of neurons and subcellular sites targeted. This study examines the cellular basis for cholinergic activation in visual cortex via M(2) type muscarinic receptors in gamma-aminobutyric acid (GABA)-ergic and non-GABAergic cells, using immunocytochemical techniques. At light microscopic resolution, M(2) immunoreactivity (-ir) was seen in all layers except area and sublayer specific bands in layer 4. Subcellularly, M(2)-ir occurred in both dendrites and terminals that form symmetric and asymmetric junctions. Layers 5 and 6 were characterized by axosomatic contacts that displayed labeling in the presynaptic component, and layer 6 displayed perikaryal postsynaptic staining, suggesting that corticofugal output neurons may be modulated particularly strongly via M(2). Infragranular layers differed from the supragranular layers in that more labeled profiles were axonal than dendritic, indicating a dominant presynaptic effect by acetylcholine via M(2) there. Unilateral cingulate cortex cuts caused reduction of cholinergic and noradrenergic fibers in the lesioned hemisphere at light microscopic resolution; at electron microscopic resolution, the synapse density and axonal M(2) labeling were reduced, suggesting that M(2) was localized presynaptically on extrathalamic modulatory inputs. Dual labeling with GABA in visual cortex layer 5 showed that half of M(2)-labeled dendrites originated from GABAergic neurons. Given that only one-fifth of all cortical dendritic profiles are GABAergic, this prevalence of dual labeling indicates an enrichment of M(2) within GABAergic dendrites and, thus, implicates abundant postsynaptic action on GABAergic neurons via M(2). In contrast, only one-tenth of M(2)-labeled terminals originated from GABAergic neurons, suggesting that the presynaptic action of acetylcholine via M(2) receptors would be more selective for non-GABAergic terminals.     

Effect of anandamide uptake inhibition in the production of nitric oxide and in the release of cytokines in astrocyte cultures

Astrocytes play a key role regulating aspects of inflammation in the central nervous system (CNS). Several enzymes, such as the inducible nitric oxide synthase (iNOS) or the cyclooxygenase-2 (COX-2), along with different inflammatory mediators such as the free radical nitric oxide (NO) or proinflammatory cytokines, have been proposed to be involved in the cell damage associated with neuroinflammation. Recent studies suggest that the endogenous cannabinoid system (ECS) may be involved in the regulation of neuroinflammation. Cannabinoid agonists decrease neurotoxicity and release of proinflammatory factors from activated glial cells and anandamide itself is able to promote antiinflammatory responses in astrocytes via CB1 cannabinoid receptors. The present study is aimed at studying whether UCM707, a potent and selective anandamide uptake inhibitor, is able to inhibit the production of proinflammatory mediators by LPS-stimulated astrocytes. Our findings indicate that UCM707 is able to reduce NO release, iNOS expression, and the production of the proinflammatory cytokines tumoral necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in a significant manner, while producing a slight increase in IL-6 levels. These effects can be reproduced by administration of the synthetic agonist HU210 and partially or totally blocked by administration of CB1 or CB2 selective antagonists, further supporting the involvement of the ECS. These results confirm the ability of UCM707 to reinforce the beneficial effects induced by anandamide and make it an attractive candidate for the management of those pathologies with neuroinflammation as one of their hallmarks.     

Interferon lambda inhibits herpes simplex virus type I infection of human astrocytes and neurons

Herpes simplex virus Type I (HSV-1) is a neurotropic virus that is capable of infecting not only neurons, but also microglia and astrocytes and can establish latent infection in the central nervous system (CNS). We investigated whether IFN lambda (IFN-λ), a newly identified member of IFN family, has the ability to inhibit HSV-1 infection of primary human astrocytes and neurons. Both astrocytes and neurons were found to be highly susceptible to HSV-1 infection. However, upon IFN-λ treatment, HSV-1 replication in both astrocytes and neurons was significantly suppressed, which was evidenced by the reduced expression of HSV-1 DNA and proteins. This IFN-λ-mediated action on HSV-1 could be partially neutralized by antibody to IFN-λ receptor. Investigation of the mechanisms showed that IFN-λ treatment of astrocytes and neurons resulted in the upregulation of endogenous IFN-α/β and several IFN-stimulated genes (ISGs). To block IFN-α/β receptor by a specific antibody could compromise the IFN-λ actions on HSV-1 inhibition and ISG induction. In addition, IFN-λ treatment induced the expression of IFN regulatory factors (IRFs) in astrocytes and neurons. Furthermore, IFN-λ treatment of astrocytes and neurons resulted in the suppression of suppressor of cytokine signaling 1 (SOCS-1), a key negative regulator of IFN pathway. These data suggest that IFN-λ possesses the anti-HSV-1 function by promoting Type I IFN-mediated innate antiviral immune response in the CNS cells.     

Roles of estrogen receptor alpha and androgen receptor in the regulation of neuronal nitric oxide synthase

In brain and peripheral tissues, steroid hormones regulate nitric oxide synthase (nNOS). We asked whether estrogen receptor-alpha (ERalpha) and/or androgen receptor (AR) regulated nNOS immunoreactivity in mouse brain. First, we quantified cells singly labeled for nNOS immunoreactivity or labeled dually with ERalpha-immunoreactive (-ir) or AR-ir cells in the nucleus accumbens (Acb), preoptic area (POA), bed nucleus of the stria terminalis (BNST), posterior dorsal and posterior ventral regions of the medial amygdala (MePD and MePV, respectively), and paraventricular nucleus (PVN). The POA and MePD contained the greatest number of double-labeled cells. More nNOS-ir cells were colabeled with ERalpha immunoreactivity compared with AR immunoreactivity. Next, by using a double mutant mouse in which males lacked functional ERalpha, AR, or both, we investigated the roles of these steroid receptors in nNOS-ir cell numbers and immunoreactive area staining under testosterone (T) and estradiol (E2) conditions. Our data show that functional ERalpha is correlated with more nNOS-ir cells under T conditions and more immunoreactive area staining in the POA under both T and E2 conditions. However, ERalpha decreases nNOS-ir cell number in the BNST under E2 treatment. In summary, the data suggest that AR has organizational actions on nNOS-ir cell numbers in the MePV, that interactions between ERalpha and AR genes occur in PVN, and that sex differences in nNOS-ir area staining are limited to the POA. Thus, we show that ERalpha and AR interact to regulate nNOS in male and female brain in a site-specific manner.     

Transforming growth factor-beta and gamma-interferon have dual effects on growth of peripheral glia

The influence of transforming growth factor-beta (TGF-beta) and gamma-interferon on DNA synthesis in Schwann cells and enteric glia in culture has been studied. TGF-beta stimulated the DNA synthesis of short-term (less than 2 weeks in culture) Schwann cells, whereas gamma-interferon was ineffective. The stimulatory effect of TGF-beta was additive to the stimulation of DNA synthesis due to axonal membrane fragments. In contrast to their effect on short-term Schwann cells, both TGF-beta and gamma-interferon inhibited DNA synthesis in enteric glial cells and in long-term (over 3 months in culture) Schwann cells. When short-term Schwann cells were stimulated to divide by axolemma or glial growth factor, gamma-interferon did not inhibit this enhanced DNA synthesis although it suppressed DNA synthesis induced by cAMP analogues. These results raise the possibility that TGF-beta and gamma-interferon might have a role in controlling glial proliferation during development and/or regeneration of the peripheral nervous system.     

Functional characterization of substance P receptors on cultured human spinal cord astrocytes: Synergism of substance P with cytokines in inducing interleukin-6 and prostaglandin E2 production

Following brain injury, astrocytes express receptors for cytokines and neuropeptides and secrete several regulatory mediators that have a well established role in inflammation, immunity, and tissue development or repair. To elucidate the role of substance P (SP), a neurotransmitter peptide of the tachykinin family, in inducing astrocyte secretory activities, we have examined the expression of SP receptors and the functional consequences of their activation in cultured astrocytes from the human embryonic brain or spinal cord. Radioligand binding studies revealed that only one type of SP receptors, the high affinity NK-1 receptor, was present on human astrocytes and that spinal cord astrocytes expressed about 6 times as many SP binding sites as brain astrocytes. Following SP treatment, a substantial inositol phosphate formation was observed in spinal cord astrocytes only. Stimulation of spinal cord astrocytes with SP alone did not induce secretion of cytokines [interleukin-6 (IL-6), granulocyte-macrophage-CSF, macrophage chemoattractant protein-1 or leukemia inhibitory factor] or prostaglandin E₂ (PGE₂). Interestingly, however, SP selectively potentiated the inducing effect of IL-1β on IL-6 and PGE₂ secretion by spinal cord astrocytes without affecting the IL-1-β-evoked secretion of other cytokines. SP also enhanced the small inducing effect of tumor necrosis factor-α (TNF-α) on IL-6 and PGE₂ secretion and that of transforming growth factor-β on PGE₂ secretion. These results suggest that SP can enhance immunoregulatory and neurotrophic astroglial functions mediated by IL-6 and PGE₂ by acting in concert with a set of cytokines whose cerebral expression has been reported during development and in a variety of diseases. GLIA 21:183–193, 1997. © 1997 Wiley-Liss, Inc.     

Selective ablation of astrocytes by intracerebral injections of α-aminoadipate

The efficacy and the specificity of the putative astrotoxin, α-aminoadipate, were examined in this study. The integrity of astrocytes was evaluated at several time points following a single injection of α-aminoadipate into amygdala of adult rats using immunohistochemistry. The density and the morphological appearance of neurons and the response of microglia were also examined. The injection of α-aminoadipate disrupted the astrocytic network in that region. There was a profound loss of glial fibrillary acidic protein-positive and S100β-positive astrocytes, normally present in the region, while vimentin immunohistochemistry revealed the presence of deformed cell processes, presumably astrocytic. The presence of reactive microglia at the injection site was suggestive of an active degenerative process, while the normal neuronal density and appearance, as compared to controls, suggested that the damage was confined to astrocytes. The confirmed effectiveness and cellular specificity of α-aminoadipate in vivo makes it a potentially important experimental tool for attempting to decipher the functional significance of astrocytes. © 1996 Wiley-Liss, Inc.     

Unusual topographical pattern of proximal astrogliosis around a cortical devascularizing lesion

Class II vessels were disrupted on the cortical surface of adult rats within a circular 5-mm-diameter area. This consistently resulted in the formation of a conical lesion by day 1, with a cystic cavity forming by day 21. Four markers were used to identify the glial response surrounding the lesion. The antibody used against S100beta marked the largest astrocytic pool in the gray matter of the cerebral cortex; only approximately 5% of astrocytes were glial fibrillary acidic protein (GFAP)(+) in control animals. GFAP served as a marker for distal reactive gliosis and vimentin (VIM) for proximal gliosis. Isolectin B4 was used as an additional marker to distinguish VIM(+) microglia from astrocytes inside the lesion area. Three immunohistochemically distinct areas of reactive astrocytes surrounding the lesion were found within 24 hr of injury and lasted through day 6. The first area, in contrast to focal traumatic injuries, consisted of a 196-microm-thick boundary layer of S100beta(+) cells immediately surrounding the lesion that never expressed GFAP or VIM by day 6. This boundary layer turns into a GFAP(+) glial limitans encasing the cystic cavity by day 21. A second unusual extended area around the base of the lesion reaching partly into the corpus callosum consisted of S100beta(+)/GFAP(+)/VIM(+) cells. This region appears to be compatible with the local or proximal gliotic response usually found completely surrounding other focal-type injuries. The proximal response at the base of the lesion developed over the first 3 days in the following sequence: S100beta(+)/GFAP(-)/VIM(-) to S100beta(+)/GFAP(+)/VIM(-) to S100beta(+)/GFAP(+)/VIM(+). Ninety percent of the astrocytes in this area express VIM. This is very high compared with findings in stab-wound preparations, where only 10% of astrocytes (surrounding entire lesion) are found to be VIM(+). A third region, consistent with a remote or distal reactive gliotic response, demonstrated staining for S100beta and had increased GFAP contents throughout the neocortical hemisphere. Cells in this region were never found to be VIM(+). Among S100beta(+) cells close to the boundary region, more than 80% expressed detectable GFAP by 2 days after lesioning. S100beta(+) cells 1 mm more laterally (distal to lesion) did not express GFAP to the same level until day 6. Thus, we find three immunohistochemically distinct populations of reactive astrocytes surrounding the focal ischemic lesion. In contrast to the case for stab-wound traumatic injury, the response closest to and surrounding the lesion did not up-regulate GFAP or VIM by day 6. The proximal response was, instead, more remote and only at the base of the lesion, extending partly into the corpus callosum.     

Regulation of protein kinases and coregulatory interplay of S-100beta and serotonin transporter on serotonin levels in diabetic rat brain

Protein kinases are critical component in the regulation of signal transduction pathways, including neurotransmitters. Our previous studies have shown that serotonin (5-HT) altered under diabetic condition was accompanied by alterations of protein kinase C-alpha (PKC-alpha) and CaMKII, and those alterations were reversed after insulin administration. The current study showed that alloxan-induced diabetic animals revealed hyperglycemia and was associated with an increase in the content of 5-HT, PKC-alpha expression and PKC activity (P < 0.05) simultaneously in striatum (ST), midbrain (MB), pons medulla (PM), cerebellum (CB), and cerebral cortex (CCX) from 7 days to 60 days. Although the 5-HT levels in hippocampus (HC) and hypothalamus (HT) were not altered, the PKC-alpha expression and PKC activity showed increases (P < 0.05) in level in HC. Insulin administration reversed all these changes to a normal level. In contrast, the in vitro study has shown that the 5-HT levels correlated with PKC-alpha expressions as well as PKC activity (P < 0.05) only in ST, MB, and CB either after induction with phorbol 12-myristate 13-acetate (PMA) or blocking with chelerythrine, whereas PM and CCX remained elevated (P < 0.05), implying a regulatory role for PKC-alpha only in ST, MB, and CB. However, our consecutive studies have shown that the 5-HT level in PM was regulated by p38-mitogen-activated protein kinase (p38-MAPK) both in vivo and in vitro, whereas the 5-HT level in CCX was coregulated by S-100beta by protein-protein interaction with serotonin transporter (SERT) via 8-bromoadenosine 3',5'-cyclic monophosphate sodium salt (8-Br-cAMP)-induced cAMP/PKAII pathway(s).     

Interleukin-1beta upregulates functional expression of neurokinin-1 receptor (NK-1R) via NF-kappaB in astrocytes

Cytokines and neuropeptides are modulators of neuroimmunoregulation in the central nervous system (CNS). The interaction of these modulators may have important implications in CNS diseases. We investigated whether interleukin-1beta (IL-1beta) modulates the expression of neurokinin-1 receptor (NK-1R), the primary receptor for substance P (SP), a potent neuropeptide in the CNS. IL-1beta upregulated NK-1R expression in human astroglioma cells (U87 MG) and primary rat astrocytes at both mRNA and protein levels. IL-1beta treatment of U87 MG cells and primary rat astrocytes led to an increase in cytosolic Ca(2+) in response to SP stimulation, indicating that IL-1beta-induced NK-1R is functional. CP-96,345, a specific non-peptide NK-1R antagonist, inhibited SP-induced rise of [Ca(2+)](i) in the astroglioma cells. Investigation of the mechanism responsible for IL-1beta action revealed that IL-1beta has the ability of activating nuclear factor-kappab (NF-kappaB). Caffeic acid phenethyl ester (CAPE), a specific inhibitor of NF-kappaB activation, not only abrogated IL-1beta-induced NF-kappaB promoter activation, but also blocked IL-1beta-mediated induction of NK-1R gene expression. These findings provide additional evidence that there is a biological interaction between IL-1beta and the neuropeptide SP in the CNS, which may have important implications in the inflammatory diseases in the CNS.