Newborn Rat Brain Research Articles

Down-regulation of mu-opioid receptor expression in rat oligodendrocytes during their development in vitro.

In the central nervous system, opioid receptors are found in neurons and also in glial cells. To gain more information on their presence and possibly on their function, we investigated the expression of mu-opioid receptors (MOR) during oligodendroglial cell development in two culture systems. In these models, during the first days, the cells are O-2A bipotential progenitor cells (also called OPCs; oligodendrocyte precursor cells), and then they differentiate into oligodendrocytes, which mature. In the first system, oligodendroglial cells, derived from newborn rat brain hemispheres, are grown in primary culture in the presence of a confluent layer of astrocytes, and they differentiate slowly. In the second, cells are specifically detached from the mixed cultures of the first system and are grown thereafter alone in secondary culture, a condition allowing a rapid cell differentiation. Under both conditions OPCs and immature oligodendrocytes were found to express a high level of MOR mRNA, whereas mature oligodendrocytes did not express it at all. The decrease of MOR expression during oligodendrocyte maturation was progressive, suggesting that it was not a primary effect of differentiation but an indirect secondary effect. Our study also shows that basic fibroblast growth factor (bFGF), which has been claimed by some authors to induce a dedifferentiation of the mature oligodendrocytes, and retinoic acid (RA), which had not been tested before, were not able to restore MOR expression in mature oligodendrocytes. These results indicate that bFGF and RA neither reverse the maturation process nor dedifferentiate the cells. However, RA was found to inhibit almost completely the expression of the myelin basic protein. The main result of this study is that MOR is expressed in progenitors and in immature oligodendrocytes, but not in mature oligodendrocytes. This suggests that MOR could be involved in some developmental process of the cells of the oligodendroglial lineage.

Neurotrophin‐3 (NT‐3) modulates early differentiation of oligodendrocytes in rat brain cortical cultures

Multiple extracellular signals are required for oligodendroglia survival, proliferation and differentiation, and increasing evidence has accumulated that also neurotrophins regulate glial cell development in the central nervous system (CNS). In the present study we have investigated the influence of neurotrophin-3 (NT-3) on the in vitro differentiation and proliferation of oligodendrocytes prepared from the brains of newborn rats. Cells were grown in chemically defined growth medium, in the absence of fetal calf serum (FCS). RT-PCR analysis confirmed the expression of mRNA encoding the NT-3 receptor trkC in oligodendrocytes throughout in vitro development. Cell morphology was observed by phase contrast microscopy and indirect immunofluorescence staining using anti-galactocerebroside (GalC) antibodies. An increase in process formation and arborization was observed 8–24 h after the treatment with NT-3 (5–50 ng/ml). Concomitantly, NT-3 caused an increase in the appearance of GalC-positive cells. Long-term treatment with NT-3 (up to seven days) did not yield any further improvement of process formation. To elucidate the molecular mechanisms and signal transduction pathways underlying the effect of NT-3 in oligodendrocytes, the time- and concentration-dependent effect of NT-3 on c-Fos protein expression was studied by Western blot analysis. The data show that NT-3 stimulated the appearance of two c-Fos immunoreactive polypeptides with apparent molecular weights of 62 and 55 kDa, respectively. This effect was maximal at a concentration of 50 ng/ml of NT-3 after 8–24 h . NT-3-modulated morphological differentiation and c-Fos protein expression was regulated by protein kinases. Whereas the protein kinase C (PKC) inhibitors staurosporine and chelerythrine chloride had a stimulatory effect on NT-3-promoted process formation, the tyrosine kinase inhibitor genistein had an inhibitory effect and mainly cells with a bipolar and immature morphology were observable. The inhibition of tyrosine kinase activity prevented NT-3-promoted induction of c-Fos protein. Thus, in addition to its mitogenic effects, NT-3 during early time points influences the in vitro differentiation of oligodendrocytes. This process involves the induction of c-Fos protein and is mediated by PKC and trosine kinase activities. GLIA 28:244–255, 1999. © 1999 Wiley-Liss, Inc.

Neurotrophin-3 (NT-3) modulates early differentiation of oligodendrocytes in rat brain cortical cultures

Multiple extracellular signals are required for oligodendroglia survival, proliferation and differentiation, and increasing evidence has accumulated that also neurotrophins regulate glial cell development in the central nervous system (CNS). In the present study we have investigated the influence of neurotrophin-3 (NT-3) on the in vitro differentiation and proliferation of oligodendrocytes prepared from the brains of newborn rats. Cells were grown in chemically defined growth medium, in the absence of fetal calf serum (FCS). RT-PCR analysis confirmed the expression of mRNA encoding the NT-3 receptor trkC in oligodendrocytes throughout in vitro development. Cell morphology was observed by phase contrast microscopy and indirect immunofluorescence staining using anti-galactocerebroside (GalC) antibodies. An increase in process formation and arborization was observed 8-24 h after the treatment with NT-3 (5-50 ng/ml). Concomitantly, NT-3 caused an increase in the appearance of GalC-positive cells. Long-term treatment with NT-3 (up to seven days) did not yield any further improvement of process formation. To elucidate the molecular mechanisms and signal transduction pathways underlying the effect of NT-3 in oligodendrocytes, the time- and concentration-dependent effect of NT-3 on c-Fos protein expression was studied by Western blot analysis. The data show that NT-3 stimulated the appearance of two c-Fos immunoreactive polypeptides with apparent molecular weights of 62 and 55 kDa, respectively. This effect was maximal at a concentration of 50 ng/ml of NT-3 after 8-24 h. NT-3-modulated morphological differentiation and c-Fos protein expression was regulated by protein kinases. Whereas the protein kinase C (PKC) inhibitors staurosporine and chelerythrine chloride had a stimulatory effect on NT-3-promoted process formation, the tyrosine kinase inhibitor genistein had an inhibitory effect and mainly cells with a bipolar and immature morphology were observable. The inhibition of tyrosine kinase activity prevented NT-3-promoted induction of c-Fos protein. Thus, in addition to its mitogenic effects, NT-3 during early time points influences the in vitro differentiation of oligodendrocytes. This process involves the induction of c-Fos protein and is mediated by PKC and trosine kinase activities.

Type 3 Iodothyronine Deiodinase Is Selectively Expressed in Areas Related to Sexual Differentiation in the Newborn Rat Brain

Type 3 Iodothyronine Deiodinase Is Selectively Expressed in Areas Related to Sexual Differentiation in the Newborn Rat Brain

Type 3 iodothyronine deiodinase is selectively expressed in areas related to sexual differentiation in the newborn rat brain.

Thyroid hormone (T4 and T3) concentrations in target tissues are greatly influenced by the activity of iodothyronine deiodinases. Type 1 and 2 deiodinases generate T3 from T4, while deiodinase type 3 (D3) transforms T4 and T3 to inactive metabolites. Coordination of the expression and activity of these enzymes is postulated to play an important role in physiology and development, making it possible that individual cells and tissues regulate the concentrations of the active hormone according to specific needs. We have analyzed the expression of D3 in the neonatal rat brain by in situ hybridization using a specific 35S-labelled riboprobe. At postnatal day 0 D3 transcripts were unexpectedly found to be selectively expressed in areas involved in sexual differentiation of the brain such as the bed nucleus of the stria terminalis and preoptic nuclei. Expression in these areas was transient and was no longer observed at postnatal day 10. These observations suggest that D3 expression is linked to the early mechanism determining sexual function and behavior.

Microglial activation by the small heat shock protein, alpha-crystallin.

Activated microglia regulate immune and inflammatory responses in the CNS under a variety of stresses due to infection, injury and disease. In this study, we show that a stress-inducible small heat shock protein, alpha-crystallin, induces in vitro activation of microglia cultured from newborn rat brain. Exposure of microglia to alpha-crystallin resulted in an increased production of nitric oxide (NO) and the expression of the inducible NO synthase (iNOS) as determined by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. Alpha-crystallin also stimulated the synthesis of the pro-inflammatory cytokine, TNF alpha. The results presented showing microglial induction of the two key immune regulatory and inflammatory molecules, i.e., NO and TNF alpha, in response to a stress-inducible protein, suggest a link between environmental stress and the CNS immune response.

The detoxification of cumene hydroperoxide by the glutathione system of cultured astroglial cells hinges on hexose availability for the regeneration of NADPH.

The ability of astroglia-rich primary cultures derived from the brains of newborn rats to detoxify exogenously applied cumene hydroperoxide (CHP) was analyzed as a model to study glutathione-mediated peroxide detoxification by astrocytes. Under the conditions used, 200 microM CHP disappeared from the incubation buffer with a half-time of approximately 10 min. The half-time of CHP in the incubation buffer was found strongly elevated (a) in cultures depleted of glutathione by a preincubation with buthionine sulfoximine, an inhibitor of glutathione synthesis, (b) in the presence of mercaptosuccinate, an inhibitor of glutathione peroxidase, and (c) in the absence of glucose, a precursor for the regeneration of NADPH. The involvement of glutathione peroxidase in the clearance of CHP was confirmed by the rapid increase in the level of GSSG after application of CHP. The restoration of the initial high ratio of GSH to GSSG depended on the presence of glucose during the incubation. The high capacity of astroglial cells to clear CHP and to restore the initial ratio of GSH to GSSG was fully maintained when glucose was replaced by mannose. In addition, fructose and galactose at least partially substituted for glucose, whereas exogenous isocitrate and malate were at best marginally able to replace glucose during peroxide detoxification and regeneration of GSH. These results demonstrate that CHP is detoxified rapidly by astroglial cells via the glutathione system. This metabolic process strongly depends on the availability of glucose or mannose as hydride donors for the regeneration of the NADPH that is required for the reduction of GSSG by glutathione reductase.

Lactate attenuates neuron specific enolase elevation in newborn rats

This study was undertaken to investigate the protective role of lactate on the hypoxic brain in newborn rats. A total of 107 7-day-old Wistar rats were divided into three groups. The lactate accumulation group was given 5% oxygen and 95% nitrogen for 30 minutes. The lactate elimination group was given 5% oxygen, a concentration of 7.5% carbon dioxide, and 87.5% nitrogen for 30 minutes. The control rats were placed in room air. Lactate levels in the brain tissue were higher in the lactate accumulation group than in those of the control group (control: 1.78 ± 0.91, lactate accumulation: 11.42 ± 1.64 mmol/kg) and significantly decreased in the lactate elimination group (4.10 ± 1.73 mmol/kg). Blood pH remained at the same levels in the two groups. Neuron specific enolase in the cerebrospinal fluid, which is the initial neurocyte damage marker, was significantly elevated in the lactate elimination group (control: 18.3 ± 7.5, lactate accumulation: 18.8 ± 7.9, lactate elimination: 63.1 ± 61.3 ng/mL). Brain adenosine 5′-triphosphate levels were significantly decreased in the lactate elimination group. Histologic findings of the brain at 72 hours after the load revealed no abnormal changes in any of the groups examined. The authors conclude that lactate accumulation plays a protective role on the hypoxic brain in newborn rats.

Common myofibroblastic features of newborn rat astrocytes and cirrhotic rat liver stellate cells in early cultures and in vivo

Double-immunolabelling techniques were employed to investigate the distribution of smooth muscle alpha-actin (actin) in glial fibrillary acidic protein (GFAP)—positive cells in rat brain during early postnatal development and maturation and in glial primary culture derived from newborn rat brain. In addition the expression of desmin was studied in the glial primary cultures as a function of the differentiation of the cells. Comparison of the cultured astroglial cells at an early age with hepatic stellate cells derived from CCl 4-induced cirrhotic rat liver, revealed features of the astrocytic cytoskeleton characteristic of myofibroblastic cells, i.e., strong expression of both myofibroblastic markers, actin and desmin. In astroglial cells with an initial morphology reminiscent of fibroblasts the non-filamentous perinuclear immunoreaction of GFAP increased with time at the expense of actin and, partially, desmin. GFAP filaments were spread throughout the cytoplasm of the cells which acquired stellate morphology. The alterations in the morphology of the cells and the distribution and intensity of staining for GFAP and actin during the differentiation of astrocytes in culture were similar to those observed in astrocytes during the maturation of the brain. In astrocytes from a newborn brain as well as in cirrhotic hepatic stellate cells, the area of immunoreaction of GFAP was reduced and confined mainly to the nuclear region. In contrast, the cells expressed actin throughout the cytoplasm. These findings may hint at a similar function of these regionally specialized perivascular myofibroblastic cells in a normal brain and diseased liver and at inverse organ-specific functions which the cells fulfill under non-pathological conditions in vivo.

Stimulation of Neonatal and Adult Brain Neurogenesis by Subcutaneous Injection of Basic Fibroblast Growth Factor

Mounting evidence indicates that extracellular factors exert proliferative effects on neurogenetic precursors in vivo. Recently we found that systemic levels of basic fibroblast growth factor (bFGF) regulate neurogenesis in the brain of newborn rats, with factors apparently crossing the blood-brain barrier (BBB) to stimulate mitosis. To determine whether peripheral bFGF affects proliferation during adulthood, we focused on regions in which neurogenesis persists into maturity, the hippocampus and the forebrain subventricular zone (SVZ). In postnatal day 1 (P1) rats, 8 hr after subcutaneous injection (5 ng/gm body weight), bFGF increased [(3)H]thymidine incorporation 70% in hippocampal and SVZ homogenates and elicited twofold increases in mitotic nuclei in the dentate gyrus and the dorsolateral SVZ, detected by bromodeoxyuridine immunohistochemistry. Because approximately 25% of proliferating hippocampal cells stimulated in vivo expressed neuronal traits in culture, bFGF-induced mitosis may reflect increased neurogenesis. bFGF effects were not restricted to the perinatal period; hippocampal DNA synthesis was stimulated by peripheral factor in older animals (P7-P21), indicating the persistence of bFGF-responsive cells and activity of peripheral bFGF into late development. To begin defining underlying mechanisms, pharmacokinetic studies were performed in P28 rats; bFGF transferred from plasma to CSF rapidly, levels rising in both compartments in parallel, indicating that peripheral factor crosses the BBB during maturity. Consequently, we tested bFGF in adults; peripheral bFGF increased the number of mitotic nuclei threefold in the SVZ and olfactory tract, regions exhibiting persistent neurogenesis. Our observations suggest that bFGF regulates ongoing neurogenesis via a unique, endocrine-like pathway, potentially coordinating neuron number and body growth, and potentially providing new approaches for treating damaged brain during development and adulthood.

Changes in the proliferative activity in the brain of offspring rats induced by the influence of131I on the maternal organism

We tested the effects of injections of small doses of131I (150 μCi) into female rats on brain development in their offspring. The brains of rats whose embryogenesis was influenced by incorporated131I were studied with the use of an immunohistochemical technique (monoclonal antibodies to nuclear antigen of cell proliferation). Single injections of131I in the above dose were shown to noticeably suppress the cell proliferative activity in a few structures of the brain of newborn rats. The most significant drop in the proliferative index in the external layers of the cerebellum was observed after131I injections performed on the 16th day of embryogenesis (when the embryonal thyroid gland begins its functioning).

Detection of hypoxic cells with the 2-nitroimidazole, EF5, correlates with early redox changes in rat brain after perinatal hypoxia–ischemia

The hypoxia-dependent activation of nitroheterocyclic drugs by cellular nitroreductases leads to the formation of intracellular adducts between the drugs and cellular macromolecules. Because this covalent binding is maximal in the absence of oxygen, detection of bound adducts provides an assay for estimating the degree of cellular hypoxia in vivo. Using a pentafluorinated derivative of etanidazole called EF5, we studied the distribution of EF5 adducts in seven-day-old rats subjected to different treatments which decrease the level of oxygen in the brain. EF5 solution was administered intraperitoneally 30 min prior to each treatment. The effect of acute and chronic hypoxia on EF5 adduct formation (binding) was studied in the brain of newborn rats exposed to global hypoxia (8% O 2 for 30, 90 or 150 min) and in the brain of chronically hypoxic rat pups with congenital cardiac defects (Wistar Kyoto). The effect of combined hypoxia–ischemia was investigated in rat pups subjected to right carotid coagulation and concurrent exposure to 8% O 2 for 30, 90 or 150 min. Brains were frozen immediately at the end of each treatment. Using a Cy3-conjugated monoclonal mouse antibody (ELK3-51) raised against EF5 adducts, hypoxic cells within brain regions were visualized by fluorescence immunocytochemistry. Brains from controls or vehicle-injected animals showed no EF5 binding. Notably, brains from animals which were chronically hypoxemic as a result of congenital cardiac defects also showed no EF5 binding. A short exposure (30 min) to hypoxia or to combined hypoxia–ischemia resulted in increased background stain and few scattered cells with low-intensity immunostaining. Acute hypoxia exposure of at least 90–150 min, which in this age animal does not result in frank cellular damage, produced patchy areas of low- to moderate-intensity fluorescence scattered throughout the brain. In contrast, 90–150 min of hypoxia–ischemia was associated with intense immunofluorescence in the hemisphere ipsilateral to the carotid occlusion, with a pattern similar to that reported previously for the histological damage seen in this model. This study provides a sensitive method for the evaluation of the level of oxygen depletion in brain tissue after neonatal hypoxia–ischemia, at times much earlier than any method demonstrates apoptotic or necrotic cell death. Since the level of in vivo formation of macromolecular adducts of EF5 depends on the degree of oxygen depletion in a tissue, intracellular EF5 binding may serve as a useful marker of regional cellular vulnerability and redox state after brain injury resulting from hypoxia–ischemia.

Role of Heme Oxygenase-1 (HO-1) in Hypoxic-Ischemic Insult of the Newborn Rat Brain

Role of Heme Oxygenase-1 (HO-1) in Hypoxic-Ischemic Insult of the Newborn Rat Brain

Increased expression of transferrin receptors and iron in amoeboid microglial cells in postnatal rats following an exposure to hypoxia

This study was aimed to ascertain the effects of hypoxia on regulation of iron in the brain of newborn rats. At 3 h and 1 day after hypoxic exposure transferrin receptor expression as detected immunohistochemically with the antibody OX-26, and the iron content as shown by Perls' staining of amoeboid microglial cells was markedly increased. The induced changes, however, were not evident at 10 min and in longer surviving rats killed at 3 and 7 days. It is suggested that the upregulation of transferrin receptor expression coupled with iron uptake by amoeboid microglial cells in the periventricular regions is a protective mechanism to facilitate the sequestration of excess iron that may have been released either from the iron-rich oligodendrocytes, or accumulated due to a disruption of its normal transportation following the hypoxic insult. This would help protect the brain from harmful effects of iron.

Distribution of pituitary adenylate cyclase activating polypeptide mRNA in the developing rat brain

PACAP is a member of the secretin/vasoactive intestinal peptide (VIP) family, isolated from hypothalamus. Recent studies have shown that PACAP is expressed in many parts of adult brain. We have studied the precise distribution of PACAP mRNA in developing rat brain, employing in situ hybridisation. PACAP mRNA is expressed in distinct parts of the embryonic rat brain from embryonic day 13, with a robust expression in developing cortex, hippocampus, amygdala and hypothalamus as well as in spinal cord and dorsal root ganglia. The expression in hippocampus and cortex diminishes towards adulthood, compared to new-born rat brain. In the mature brain, PACAP mRNA is located in alternating layers of cerebral cortex (layers I, III and V), in the dentate gyrus, in CA4 and CA1 regions, but not in CA2 or CA3 of the hippocampus. The presence of PACAP mRNA in different structures of developing rat brain suggests an important function for this peptide during brain development.

Effect of ketamine on hypoxic–ischemic brain damage in newborn rats

The present study tests the hypothesis that ketamine, a dissociative anesthetic known to be a non-competitive antagonist of the NMDA receptor, will attenuate hypoxic–ischemic damage in neonatal rat brain. Studies were performed in 7-day-old rat pups which were divided into four groups. Animals of the first group, neither ligated nor exposed to hypoxia, served as controls. The second group was exposed to hypoxic–ischemic conditions and sacrificed immediately afterwards. Animals of the third and fourth groups were treated either with saline or ketamine (20 mg/kg, i.p.) in four doses following hypoxia. Hypoxic–ischemic injury to the left cerebral hemisphere was induced by ligation of the left common carotid artery followed by 1 h of hypoxia with 8% oxygen. Measurements of high energy phosphates (ATP and phosphocreatine) and amino acids (glutamate and glutamine) and neuropathological evaluation of the hippocampal formation were used to assess the effects of hypoxia–ischemia. The combination of common carotid artery ligation and exposure to an hypoxic environment caused major alterations in the ipsilateral hemisphere. In contrast, minor alterations in amino acid concentrations were observed after the end of hypoxia in the contralateral hemisphere. These alterations were restored during the early recovery period. Post-treatment with ketamine was associated with partial restoration of energy stores and amino acid content of the left cerebral hemisphere. Limited attenuation of the damage to the hippocampal formation as demonstrated by a reduction in the number of damaged neurons was also observed. These findings demonstrate that systemically administered ketamine after hypoxia offers partial protection to the newborn rat brain against hypoxic–ischemic injury.

Angiotensin II type 2 receptor-mediated apoptosis of cultured neurons from newborn rat brain.

Angiotensin II (Ang II) type 2 (AT2) receptors are highly expressed in neonate brain and may have a role in developmental processes such as apoptosis. Concurrent activation of c-Jun N-terminal kinase (JNK) and inhibition of Erk mitogen-activated protein kinase activities is important for apoptosis in many cells, and we previously demonstrated that stimulation of AT2 receptors causes decreased mitogen-activated protein kinase activity in neurons cultured from newborn rat hypothalamus and brain stem. Using such cultures we have employed terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling and internucleosomal DNA fragmentation to assess the role of AT2 receptors in neuronal apoptosis. Ang II (100 nM; 4-72 h) alone produced no significant neuronal apoptosis, and AT2 receptor activation did not stimulate JNK activity. However, exposure of cultures to UV radiation (6 J/m2/sec for 4 sec) to stimulate JNK elicited neuronal apoptosis that was significantly enhanced by Ang II, an effect that was abolished by the AT2 receptor antagonist PD 123,319 (1 microM) or the serine/threonine phosphatase inhibitor okadaic acid (3 nM). Additionally, Ang II enhanced the UV radiation-induced decrease in the levels of the DNA repair enzyme poly-(ADP-ribose) polymerase. These data indicate that Ang II, via AT2 receptors and activation of a serine/threonine phosphatase, contributes to neuronal apoptosis.

Angiotensin II Type 2 Receptor-Mediated Apoptosis of Cultured Neurons from Newborn Rat Brain

Angiotensin II Type 2 Receptor-Mediated Apoptosis of Cultured Neurons from Newborn Rat Brain

Neurotrophins regulate the function of cultured microglia.

Although the physiological role of neurotrophins in neuronal development and survival has been extensively investigated, their role in glial cell physiology remains to be elucidated. In the present study, we investigated the effects of neurotrophins on cultured microglia from newborn rat brain. All of the neurotrophins tested nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4), increased the secretion of plasminogen and urokinase type-plasminogen activator and specific activity of acid phosphatase, but suppressed the release of constitutively-produced and lipopolysaccharide-stimulated nitric oxide (NO) from microglia. The reverse transcription-polymerase chain reaction, immunocytochemical staining, and Western blotting revealed that cultured microglia express Trk A, B, and C, and low-affinity NGF receptor, LNGFRp75. Neurotrophin was found to phosphorylate Trk A and B, and the neurotrophin-induced enhancement of plasminogen-secretion was suppressed by protein kinase inhibitor, K252a. Furthermore, neurotrophins caused an activation of transcription factor, NF-kappaB. These results indicate that the neurotrophin family regulate the function of microglia through Trk and/or LNGFRp75-mediated signal transduction.

Microglia generate external proton and potassium ion gradients utilizing a member of the H/K ATPase family

An ion selective electrode in self-referencing mode was employed to detect ionic concentration gradients at the vicinity of microglia isolated from newborn rat brains. At 5 mM extracellular potassium concentration, a gradient of -9.43+/-4.2 microM (n=48) was recorded dissipating over a distance of 10 microm from the outer surface of the cell membrane. Pharmacological studies indicated that neither the Na+/K+-ATPase nor the inward rectifier potassium channel makes significant contributions to generation of this gradient. The recorded potassium gradient was found to be augmented by increase in extracellular potassium or proton concentrations and could be inhibited by Omeprazole (10 microM) and by the specific H+/K+-ATPase blocker SCH28080 (1 microM). These, along with the coexistence of a gradient of excess of protons, strongly suggest that a K/H ATPase is the major generator of both the potassium and the proton gradients. The Kd of the glial transporter for K ions is an order of magnitude higher (3.7 mM) than that of the epithelial H+/K+-ATPase. This is a first report of an H+/K+ transporter in microglia cells with a Kd in the physiological range of [K+]out. Implications of the H+/K+-ATPase on potassium homeostasis in microglia under high extracellular potassium and low pH, as found at the site of brain injury, are discussed.