Brain injury in a dish: a model for reactive gliosis.

Reactive gliosis is a powerful response to brain injury and subsequent neuronal damage in vivo. Neuronal cell cultures are now well established as assays to study this process in vitro. However, equivalent studies of purified glial cell populations have only recently been achieved, following the realization that glial cells produce many of the neuropeptides, transmitters and growth factors that are produced also by neurons. There is now scope for studies in vitro that use mixed, identified populations of glial and neuronal cells to dissect the interactions between the two. Such cultures also lend themselves to assays for potential therapeutic strategies for brain injury that take account of all the different cell types found in the brain.

Implications of prolonged expression of Fos-related antigens.

The AP-1 transcription factors are composed of the Fos and Fos-related antigens as well as Jun and related proteins. These factors have been extensively studied in many diverse paradigms using acute stimuli. Recent attention has focussed on long-term elevation of Fos-related antigens in the CNS, and this is discussed by Keith Pennypacker, Jau-S. Hong and Michael McMillian. Repeated or chronic treatment elevates Fos-related antigen levels for days in many different brain regions. Both direct and indirect stimulation are responsible for the protracted increase in Fos-related antigen-immunoreactive proteins, which may modulate late onset genes involved in neuroplasticity. Understanding the role of these factors in long-lasting or permanent disease states may provide insight into potential therapeutic strategies to treat chronic CNS disorders.

Effects of muscarinic, alpha-adrenergic, and substance P agonists and ionomycin on ion transport mechanisms in the rat parotid acinar cell. The dependence of ion transport on intracellular calcium.

The relationship between receptor-mediated increases in the intracellular free calcium concentration [( Ca]i) and the stimulation of ion fluxes involved in fluid secretion was examined in the rat parotid acinar cell. Agonist-induced increases in [Ca]i caused the rapid net loss of up to 50-60% of the total content of intracellular chloride (Cli) and potassium (Ki), which is consistent with the activation of calcium-sensitive chloride and potassium channels. These ion movements were accompanied by a 25% reduction in the intracellular volume. The relative magnitudes of the losses of Ki and the net potassium fluxes promoted by carbachol (a muscarinic agonist), phenylephrine (an alpha-adrenergic agonist), and substance P were very similar to their characteristic effects on elevating [Ca]i. Carbachol stimulated the loss of Ki through multiple efflux pathways, including the large-conductance Ca-activated K channel. Carbachol and substance P increased the levels of intracellular sodium (Nai) to more than 2.5 times the normal level by stimulating the net uptake of sodium through multiple pathways; Na-K-2Cl cotransport accounted for greater than 50% of the influx, and approximately 20% was via Na-H exchange, which led to a net alkalinization of the cells. Ionomycin stimulated similar fluxes through these two pathways, but also promoted sodium influx through an additional pathway which was nearly equivalent in magnitude to the combined uptake through the other two pathways. The carbachol-induced increase in Nai and decrease in Ki stimulated the activity of the sodium pump, measured by the ouabain-sensitive rate of oxygen consumption, to nearly maximal levels. In the absence of extracellular calcium or in cells loaded with the calcium chelator BAPTA (bis[o-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid) the magnitudes of agonist- or ionomycin-stimulated ion fluxes were greatly reduced. The parotid cells displayed a marked desensitization to substance P; within 10 min the elevation of [Ca]i and alterations in Ki, Nai, and cell volume spontaneously returned to near baseline levels. In addition to quantitating the activation of various ion flux pathways in the rat parotid acinar cell, these results demonstrate that the activation of ion transport systems responsible for fluid secretion in this tissue is closely linked to the elevation of [Ca]i.

Effects of extracellular ATP on ion transport systems and [Ca2+]i in rat parotid acinar cells. Comparison with the muscarinic agonist carbachol.

The effects of extracellular ATP on ion fluxes and the intracellular free Ca2+ concentration ([Ca2+]i) were examined using a suspension of rat parotid acinar cells and were contrasted with the effects of the muscarinic agonist carbachol. Although ATP and carbachol both rapidly increased [Ca2+]i about threefold above the resting level (200-250 nM), the effect of ATP was due primarily to an influx of Ca2+ across the plasma membrane, while the initial response to carbachol was due to a release of Ca2+ from intracellular stores. Within 10 s, ATP (1 mM) and carbachol (20 microM) reduced the cellular Cl- content by 39-50% and cell volume by 15-25%. Both stimuli reduced the cytosolic K+ content by 57-65%, but there were marked differences in the rate and pattern of net K+ movement as well as the effects of K+ channel inhibitors on the effluxes initiated by the two stimuli. The maximum rate of the ATP-stimulated K+ efflux (approximately 2,200 nmol K+/mg protein per min) was about two-thirds that of the carbachol-initiated efflux rate, and was reduced by approximately 30% (vs. 60% for the carbachol-stimulated K+ efflux) by TEA (tetraethylammonium), an inhibitor of the large conductance (BK) K+ channel. Charybdotoxin, another K+ channel blocker, was markedly more effective than TEA on the effects of both agonists, and reduced the rate of K+ efflux initiated by both ATP and carbachol by approximately 80%. The removal of extracellular Ca2+ reduced the ATP- and the carbachol-stimulated rates of K+ efflux by 55 and 17%, respectively. The rate of K+ efflux initiated by either agonist was reduced by 78-95% in cells that were loaded with BAPTA to slow the elevation of [Ca2+]i. These results indicated that ATP and carbachol stimulated the efflux of K+ through multiple types of K(+)-permeable channels, and demonstrated that the relative proportion of efflux through the different pathways was different for the two stimuli. ATP and carbachol also stimulated the rapid entry of Na+ into the parotid cell, and elevated the intracellular Na+ content to 4.4 and 2.6 times the normal level, respectively. The rate of Na+ entry through Na(+)-K(+)-2Cl- cotransport and Na(+)-H+ exchange was similar whether stimulated by ATP, carbachol, or ionomycin, and uptake through these two carrier-mediated transporters accounted for 50% of the ATP-promoted Na+ influx. The remainder may be due to a nonselective cation channel and an ATP-gated cation channel that is also permeable to Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Apolipoprotein E and mimetic peptide initiate a calcium-dependent signaling response in macrophages.

Apolipoprotein E (ApoE) is a 34-kDa cholesterol transport protein that also possesses immunomodulatory properties. In this study, we demonstrate that ApoE initiates a signaling cascade in murine peritoneal macrophages that leads to increased production of inositol triphosphate with mobilization of intracellular Ca(2+) stores. This cascade is inhibited by pretreatment with receptor-associated protein and Ni(2+), and it is mediated by a pertussis toxin-sensitive G protein. These properties are characteristic of signal transduction induced via ligand binding to the cellular receptor, lipoprotein receptor-related protein. A peptide derived from the receptor-binding region of ApoE also initiates signal transduction in a manner similar to that of the intact protein, suggesting that this isolated region is sufficient for signal transduction. The ApoE-mimetic peptide competed for binding with the intact protein, confirming that they both interact with the same site. ApoE-dependent signal transduction might play a role in mediating the functional properties of this lipoprotein.

Role of protein kinase C in microglia-induced neurotoxicity in mesencephalic cultures.

Microglial activation selectively kills certain neuron populations in mixed neuronal/glial cultures, which may prove useful for modeling neurodegenerative diseases such as Parkinson's disease. In mesencephalic mixed neuronal/glial cultures, microglial activation by zymosan A killed more dopaminergic neurons, assessed by [3H]dopamine uptake and by counting tyrosine hydroxylase-immunoreactive neuron number, than did microglial activation by lipopolysaccharide (LPS). The additional toxicity of zymosan resulted from microglial protein kinase C (PKC) activation. Both zymosan and PMA, but not LPS, activated PKC in enriched microglial preparations. In the mixed neuronal/glial cultures, activation of PKC by phorbol myristate acetate (PMA) increased LPS-induced nitric oxide (NO; by nitrite measurements), but not zymosan-induced NO production, and increased LPS-induced dopaminergic neurotoxicity, but not zymosan-induced dopaminergic neurotoxicity. Additive effects of PMA and LPS, similar to zymosan effects alone, reflected activation of distinct neurotoxic pathways in the microglia. The NO synthase inhibitor N-nitro-L-arginine methyl ester (NAME) totally blocked the neurotoxicity of LPS, and partially blocked zymosan-induced neurotoxicity; NAME did not block the PKC component of neurotoxicity. In addition to stimulating NO production as effectively as LPS, zymosan also activates microglial PKC and associated non-NO-mediated neurotoxic pathways that may be important in human neurodegenerative diseases. Since the role of NO in human microglia-induced neurotoxicity is controversial, zymosan may prove more useful than LPS as a microglial activator in the rodent mixed neuronal/glial culture model.

Modulatory effects of [Met5]-enkephalin on interleukin-1 beta secretion from microglia in mixed brain cell cultures.

In the present study, functional interactions between [Met5]-enkephalin (ME), naloxone and lipopolysaccharide (LPS) on interleukin-1 beta (IL-1 beta) immunostaining and secretion have been assessed in mixed brain cell cultures from embryonic day 17 mice. Adding ME alone or together with LPS to the culture increased the release of IL-1 beta after 48 h in a concentration-dependent fashion. In situ hybridization studies showed that LPS, but not ME, increased the abundance of IL-1 beta mRNA. The enhanced release of IL-1 beta caused by ME or LPS was partially blocked by naloxone. LPS induced concentration-dependent morphological changes in microglia in mixed brain cell cultures, identified by a monoclonal antibody F4/80 which is specific for macrophages/microglia. Despite increasing IL-1 beta release into the media, ME (10(-8) M) did not induce morphological changes in microglia. Naloxone alone also had no effect on glial morphology; however, the LPS-induced morphological changes were blocked by naloxone. Our data indicate that both exogenous and endogenous opioids regulate IL-1 beta production by microglial cells in the mixed brain cell cultures.

Two distinct cytosolic calcium responses to extracellular ATP in rat parotid acinar cells.

1. Increasing concentrations of ATP (0.5 microM-300 microM) produced a biphasic increase in intracellular calcium concentration [Ca]i in rat parotid acinar cells, reflecting two distinct Cai responses to extracellular ATP. 2. In the absence of Mg2+ (with 3 mM CaCl2 in the buffer solution), the more sensitive response was maximal at 3-5 microM and was not further increased by 30 microM ATP. This response to ATP was not well maintained and was blocked by ADP (0.5 mM). A second, much larger increase in Cai was observed on addition of 300 microM ATP. This larger effect, which we have described previously, appears to be mediated by ATP4-, and was selectively reversed by 4,4'-di-isothiocyanato-dihydrostilbene-2,2'-disulphonate as well as by high concentrations of alpha,beta-methylene ATP. 3. Among ATP analogues, only the putative P2Z agonist, 3'-0-(4-benzoyl)benzoyl-ATP distinguished between the two responses. This analogue was at least 10 fold more potent than ATP in stimulating the ATP(4-)-response, but did not evoke the more sensitive response. The agonist potency series for both responses to ATP was identical for other analogues examined (ATP > ATP gamma S = 2-methylthio ATP (a P2y-selective agonist) >> ADP, ITP and alpha,beta-methylene ATP (a P2x-selective agonist)). 4. Although the effect of ATP4- could best be characterized as a P2z-type purinoceptor response, this effect was strongly and selectively blocked by reactive blue 2, a putative P2y-purinoceptor antagonist. Reactive blue 2 may bind to and block P2z purinoceptors since [gamma 32P]-ATP binding to parotid cells was inhibited by this compound. 5. In contrast to the response to ATP4-, the more sensitive response to ATP was potentiated by 2+ reactive blue 2 and was less affected by increases in external Mg2+ and Ca2+.6. Parasympathetic denervation selectively increased the more sensitive response, suggesting that it maybe physiologically regulated.

Regulation of preproenkephalin expression in astrocytes: is there a role for glia-derived opioid peptides in reactive gliosis?

Cultured glial cells synthesize a number of neuropeptides, growth factors, cytokines, and other factors that are not detectable in resting glia in the adult brain but that appear in response to brain injury. Enkephalin-containing peptides are produced and released by cultured astrocytes and glioma cell lines, which have provided convenient model systems to study the regulation of the preproenkephalin gene. The purpose of this review is to examine data suggesting that reactive astrocytes produce enkephalin-containing peptides, the regulatory processes that may be involved, and possible functions of glia-derived peptides in reactive gliosis.

Blockade of ATP binding site of P2 purinoceptors in rat parotid acinar cells by isothiocyanate compounds.

Extracellular ATP activates a P2Z-type purinergic receptor (purinoceptor) in rat parotid acinar cells that increases the intracellular free Ca2+ concentration via the entry of extracellular Ca2+ through an ATP-sensitive cation channel (Soltoff et al., Am J Physiol 262: C934-C940, 1992). To learn more about the ATP binding site of the purinoceptor, we examined the effects of several stilbene isothiocyanate analogs of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), which block the binding of [32P]ATP to intact parotid cells (McMillian et al., Biochem J 255:291-300, 1988) and blocked the activation of the P2Z purinoceptor. The ATP-stimulated 45Ca2+ uptake was blocked by DIDS, H2DIDS (dihydro-DIDS; 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid), and SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid), but not by DNDS (4,4'-dinitrostilbene-2,2'-disulfonic acid), a stilbene disulfonate compound lacking isothiocyanate (SCN-) groups, or by KSCN. The potency of the stilbene disulfonates was related to the number of isothiocyanate groups on each compound. Under the experimental conditions, the IC50 value of DIDS (approximately 35 microM), which has two SCN-groups, was much lower than that of SITS (approximately 125 microM), which has only one SCN-group. The inhibitory effects of DIDS appeared to be much more potent than those of SITS due to the kinetics of their binding to the purinoceptors. Eosin-5-isothiocyanate (EITC) and fluorescein-5-isothiocyanate (FITC), non-stilbene isothiocyanate compounds with single SCN-groups, also blocked the response to ATP and were less potent than DIDS. Trinitrophenyl-ATP (TNP-ATP), an ATP derivative that is not an effective agonist of the parotid P2Z receptor, blocked the covalent binding of DIDS to the plasma membrane, suggesting that ATP and DIDS bind to the same site. Reactive Blue 2 (Cibacron Blue 3GA), an anthraquinone-sulfonic acid derivative that is a noncovalent purinergic antagonist, also blocked the covalent binding of DIDS to the plasma membrane. These results suggest that isothiocyanate compounds interact with the ATP binding site of this P2 purinoceptor, and that isothiocyanate groups make an important contribution in determining the effectiveness of the stilbene disulfonate compounds in blocking the binding of nucleotide agonists to this purinoceptor.

Protection against glutamate toxicity through inhibition of the p44/42 mitogen-activated protein kinase pathway in neuronally differentiated P19 cells.

Excessive levels of the neurotransmitter glutamate trigger excitotoxic processes in neurons that lead to cell death. N-Methyl-D-aspartate (NMDA) receptor over-activation is a key excitotoxic stimulus that leads to increases in intracellular calcium and activation of downstream signaling pathways, including the p44/42 mitogen-activated protein (MAP) kinase pathway. In the present study, we have demonstrated that 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a potent and selective inhibitor of the p44/42 MAP kinase signaling pathway, prevents glutamate-induced death in neuronally differentiated P19 cells. In addition, we show that differentiated, but not undifferentiated, P19 cells expressed zeta1, epsilon1, and epsilon2 subunits of the NMDA receptor. Differentiated P19 cells exhibited specific NMDA receptor binding and intracellular calcium responses to glutamate that were blocked by the selective NMDA receptor antagonist [5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), but not U0126. Glutamate treatment of differentiated P19 cells triggered a rapid and sustained induction in p42 MAP kinase phosphorylation that was blocked by U0126. Pretreatment of differentiated P19 cells with U0126, but not other classes of protein kinase inhibitors, protected against glutamate-induced cell death. Post-treatment with U0126, even as late as 6 hr after glutamate application, also protected against glutamate toxicity. These results suggest that the p44/42 MAP kinase pathway may be a critical downstream signaling pathway in glutamate receptor-activated toxicity.

Elevated basal AP-1 DNA binding activity in developing rat brain.

Nuclear extracts from hippocampi, striata and hypothalami of postnatal day (P) 7 rats contained elevated basal levels of AP-1 DNA binding activity and c-jun protein, which decreased to the low basal levels observed in the adult by P21. In contrast to the AP-1 DNA binding complex in the adult brain, the fos-related antigens were not a major component of the P7 AP-1 DNA binding activity.

Transcription factors in primary glial cultures: changes with neuronal interactions.

Several astrocyte gene products, such as enkephalin and glial fibrillary acidic protein (GFAP), are expressed at higher levels under in vitro conditions relative to in vivo. We have observed that cultured glial cells express high basal levels of transcription factors, such as fos-related antigens (Fra), c-Jun, JunD, and cAMP responsive element binding protein (CREB). When neuronal cells are plated on top of the monolayers, the expression of Fra, c-Jun, JunD, and GFAP decreases in the astroglial cells. The DNA binding activity to the AP-1-like sites of the GFAP and proenkephalin genes was examined in these cultures. The protein complex from glial cultures which recognizes the GFAP AP-1 element contained Fra immunoreactivity while the DNA binding from mixed neuronal/glial cultures consists of CREB-immunoreactive proteins. In glial cultures, no binding occurred to the proenkephalin AP-1-like element but a CREB-immunoreactive complex recognized this sequence in the mixed cultures. Thus, with the addition of neurons, both transcription factors and target gene products decrease in astroglial cells. The proteins that compose gene modulatory complexes also change suggesting that regulation of astroglial gene expression is modulated by neurons.

Basal expression of 35 kDa fos-related antigen in olfactory bulb.

Recently, there have been a number of reports showing a long-term increased expression of fos-related antigens (fra), molecular weight of 35 kDa, after brain injury or chronic treatment of rats with various drugs. We report elevated basal levels of this transcription factor in the olfactory bulb relative to other brain regions. The expression of this protein is further enhanced in the olfactory bulb as long as 3 months after a single injection of kainate, an effect similar to that we previously observed in the hippocampus. The AP-1 DNA binding activity in olfactory bulb from kainate-treated rats contains fra and jun immunoreactivity suggesting that the 35 kDa fra dimerizes with jun protein, probably junD, to bind to AP-1 sites. Elevated basal levels of this transcription factor in the olfactory bulb appear to be related to the constant reinnervation and synaptogenesis which occurs in this brain region. The 35 kDa fra may be involved in long-term genomic program changes required to adapt to an altered biochemical environment.

Acute repeated nicotine injections increase enkephalin and decrease AP-1 DNA binding activity in rat adrenal medulla.

Previously we reported that a single injection of nicotine decreased AP-1 DNA binding activity in adrenal medullae, although chronic bidaily nicotine (and saline) injections increased this binding activity [15]. Repeated acute nicotine injections (3 mg/kg i.p., 7 injections equi-spaced over a 3 h period) effectively increased adrenal tyrosine hydroxylase [3] and [Met5]enkephalin levels and also profoundly decreased adrenal medulla AP-1 DNA binding activity for over 8 h.

Elevation of [Ca2+]i and the activation of ion channels and fluxes by extracellular ATP and phospholipase C-linked agonists in rat parotid acinar cells.

Extracellular ATP initiates a variety of changes in the parotid acinar cell. The initial effect appears to be the entry of Ca2+ (and perhaps Na+), and a series of ion transport events result from the subsequent elevation of [Ca2+]i. Agonists of phospholipase C-linked receptors elevate [Ca2+]i by a different pathway, involving the generation of inositol polyphosphate compounds, but share in the subsequent initiation of the ion transport events. Although the maintenance of the physiological changes may depend on specific inositol polyphosphate intermediates, the critical initiating factor is the elevation of [Ca2+]i. Fluid secretion by the parotid gland is triggered by the action of neurotransmitters, which alter the membrane permeability of the acinar cell. The similarities between the two receptor-mediated activation pathways suggests that ATP may act as a neurotransmitter and play a role in the control of fluid secretion. Basing our analysis on the purinoceptor characteristics outlined by Gordon, we suggest that the parotid receptor belongs to the P2Z class, which is highly sensitive to ATP4-. Basing his analysis on the earlier report by Gallacher of the effects of ATP on mouse parotid cells, Gordon placed the parotid purinoceptor in a different P2 subclass (P2Y). However, our findings of an increased potency of ATP in the absence of Mg2+, as well as the potency order of different nucleotides, indicate that the P2Z class is a more appropriate category.

Endogenous apolipoprotein E suppresses LPS-stimulated microglial nitric oxide production.

The human apolipoprotein (apo) E4 isoform is associated with an increased risk for Alzheimer's disease (AD) and poor prognosis after acute CNS injury. Addition of human apoE inhibits murine microglial activation in culture, suggesting that microglia might be an important physiological target of apoE. In the present study, we examined the role of endogenous murine apoE in modulating microglial nitric oxide (NO) production following lipopolysaccharide (LPS) stimulation. Brain cultures from apoE-deficient mouse pups showed enhanced NO production relative to cultures from wild-type mice and from transgenic mice expressing the human apoE3 isoform, demonstrating that endogenous apoE produced by glial cultures is capable of inhibiting microglial function. ApoE produced within the brain may suppress microglial reactivity and thus alter the CNS response to acute and chronic injury.

Parasympathetic denervation increases responses to VIP in isolated rat parotid acini.

Vasoactive intestinal peptide (VIP) is a putative neurotransmitter found in the salivary glands of many species, including the rat parotid gland. Parasympathetic denervation has been reported to deplete VIP in the rat parotid gland and to lead to supersensitivity to this peptide in vivo. We have compared the effects of VIP on acini isolated from parasympathetically denervated and unoperated parotid glands to examine possible supersensitivity to the peptide in vitro. VIP normally produced responses similar to those obtained with a low concentration of the beta adrenergic agonist isoproterenol (ISO), but strikingly different from the effects obtained with the muscarinic agonist carbachol (CARB). In parotid membrane preparations, VIP stimulated adenylate cyclase activity. Dissociated acini treated with VIP showed increases in cAMP accumulation and amylase release which were potentiated by forskolin and also by inhibition of phosphodiesterase. After parasympathetic denervation, maximal effects of VIP on adenylate cyclase, cAMP accumulation and amylase release in intact cells were increased two- to five-fold over contralateral control (or unoperated) parotid responses. The increase in adenylate cyclase-mediated responses after denervation was specific to VIP; there was no increased response nor increased sensitivity of any of these responses to ISO. Specific [125I]VIP binding to parotid acini increased two-fold per gland and three-fold per mg of protein after denervation; this probably explains the observed increases in the response to VIP.

Lack of desensitization to neurokinin A due to partial agonist property of this peptide at parotid substance P receptors.

Effects of substance P (SP) and neurokinin A (NKA) on intracellular free calcium concentration (Cai) were compared in Fura2-loaded parotid acinar cells. SP produced a pronounced increase in Cai which rapidly desensitized. In contrast, NKA produced a smaller increase which was well maintained. Cells desensitized to SP were cross-desensitized to NKA. The absence of NKA-induced desensitization reflects the inability of NKA to fully activate parotid SP receptors; NKA is a partial agonist in the parotid. Similarly, the action of NKA as a partial agonist in other systems may explain findings that SP responses desensitize while NKA responses do not.

Protein tyrosine kinase inhibitors suppress the production of nitric oxide in mixed glia, microglia-enriched or astrocyte-enriched cultures.

Nitric oxide (NO) produced by glial cells has been implicated in the neuropathogenesis of various diseases. However, the signaling transduction pathway(s) for the production of NO in these cells is not well understood. To test whether protein tyrosine kinases (PTKs) are required for signaling events of NO production in glial cells, this study examined the effects of genistein and tyrphostin A25, two potent inhibitors of PTKs, on the production of NO in mouse primary mixed glia, microglia-enriched or astrocyte-enriched cultures exposed to lipopolysaccharide (LPS) or a combination of LPS and interferon-gamma (IFN gamma). LPS induced a dose-dependent increase in NO production from the mixed glia cultures. The LPS-induced NO production was significantly enhanced by stimulating the cells with IFN gamma. Genistein or tyrphostin A25 inhibited the production of NO in both LPS- and IFN gamma/LPS-stimulated mixed glia cultures. The production of NO in the stimulated microglia-enriched or astrocyte-enriched cultures was also inhibited by tyrphostin A25. To verify the cellular sources of NO, immunocytochemical staining of inducible NO synthase (iNOS) was followed by staining with the microglia marker Mac-1 or the astrocyte marker glial fibrillary acid protein (GFAP) in microglia-enriched or astrocyte-enriched cultures. The expression of iNOS and the production of NO in microglia-enriched cultures were significantly higher than those in the identically stimulated astrocyte-enriched cultures. These results demonstrate that PTKs are involved in the signaling events of LPS-induced NO production in microglia and astrocytes, and that microglia are more responsive than astrocytes to stimuli which induce NO. These results may provide insights into therapeutic interventions in the pathway for NO production in the brain.