Neonatal Rat Astrocytes Research Articles

Pyruvate dehydrogenase kinase 2 knockdown restores the ability of amyotrophic lateral sclerosis-linked SOD1G93A rat astrocytes to support motor neuron survival by increasing mitochondrial respiration.

Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron (MN) degeneration. Various studies using cellular and animal models of ALS indicate that there is a complex interplay between MN and neighboring non-neuronal cells, such as astrocytes, resulting in noncell autonomous neurodegeneration. Astrocytes in ALS exhibit a lower ability to support MN survival than nondisease-associated ones, which is strongly correlated with low-mitochondrial respiratory activity. Indeed, pharmacological inhibition of pyruvate dehydrogenase kinase (PDK) led to an increase in the mitochondrial oxidative phosphorylation pathway as the primary source of cell energy in SOD1G93A astrocytes and restored the survival of MN. Among the four PDK isoforms, PDK2 is ubiquitously expressed in astrocytes and presents low expression levels in neurons. Herein, we hypothesize whether selective knockdown of PDK2 in astrocytes may increase mitochondrial activity and, in turn, reduce SOD1G93A-associated toxicity. To assess this, cultured neonatal SOD1G93A rat astrocytes were incubated with specific PDK2 siRNA. This treatment resulted in a reduction of the enzyme expression with a concomitant decrease in the phosphorylation rate of the pyruvate dehydrogenase complex. In addition, PDK2-silenced SOD1G93A astrocytes exhibited restored mitochondrial bioenergetics parameters, adopting a more complex mitochondrial network. This treatment also decreased lipid droplet content in SOD1G93A astrocytes, suggesting a switch in energetic metabolism. Significantly, PDK2 knockdown increased the ability of SOD1G93A astrocytes to support MN survival, further supporting the major role of astrocyte mitochondrial respiratory activity in astrocyte-MN interactions. These results suggest that PDK2 silencing could be a cell-specific therapeutic tool to slow the progression of ALS.

Cytotoxic Effects of Cannabidiol on Neonatal Rat Cortical Neurons and Astrocytes: Potential Danger to Brain Development.

The influence of cannabidiol (CBD) on brain development is inadequately understood. Since CBD is considered a non-intoxicating drug, it has attracted great interest concerning its potential medical applicability, including in pregnant women and children. Here, we elucidated the response of perinatal rat cortical neurons and astrocytes to CBD at submicromolar (0.1, 0.5, 1, 5 µM) concentrations attainable in humans. The effect of CBD was concentration- and time-dependent and cell-specific. In neurons, 0.1 µM CBD induced an early and transient change in mitochondrial membrane potential (ΔΨm), ATP depletion, and caspase-8 activation, followed by rapid ATP recovery and progressive activation of caspase-9 and caspase-3/7, resulting in early apoptotic cell death with reduction and shortening of dendrites, cell shrinkage, and chromatin condensation. The decrease in neuronal viability, ATP depletion, and caspase activation due to CBD exposure was prevented by transient receptor potential vanilloid 1 (TRPV1) antagonist. In astrocytes, 0.5 µM CBD caused an immediate short-term dysregulation of ΔΨm, followed by ATP depletion with transient activation of caspase-8 and progressive activation of caspase-9 and caspase-3/7, leading to early apoptosis and subsequent necroptosis. In astrocytes, both TRPV1 and cannabinoid receptor 1 (CB1) antagonists protected viability and prevented apoptosis. Given that CBD is a non-intoxicating drug, our results clearly show that this is not the case during critical periods of brain development when it can significantly interfere with the endogenous cannabinoid system.

The uptake of [3H]dopamine into cultured neonatal rat astrocytes involves multiple transporters

The uptake of [3H]dopamine into cultured neonatal rat astrocytes involves multiple transporters

Astrocytic responses to high glucose impair barrier formation in cerebral microvessel endothelial cells.

Hyperglycemic conditions are prodromal to blood-brain barrier (BBB) impairment. The BBB comprises cerebral microvessel endothelial cells (CMECs) that are surrounded by astrocytic foot processes. Astrocytes express high levels of gap junction connexin 43 (Cx43), which play an important role in autocrine and paracrine signaling interactions that mediate gliovascular cross talk through secreted products. One of the key factors of the astrocytic "secretome" is vascular endothelial growth factor (VEGF), a potent angiogenic factor that can disrupt BBB integrity. We hypothesize that high-glucose conditions change the astrocytic expression of Cx43 and increase VEGF secretion leading to impairment of CMEC barrier properties in vitro and in vivo. Using coculture of neonatal rat astrocytes and CMEC, we mimic hyperglycemic conditions using high-glucose (HG) feeding media and show a significant decrease in Cx43 expression and the corresponding increase in secreted VEGF. This result was confirmed by the analyses of Cx43 and VEGF protein levels in the brain cortex samples from the type 2 diabetic rat (T2DN). To further characterize inducible changes in BBB, we measured transendothelial cell electrical resistance (TEER) and tight junction protein levels in cocultured conditioned astrocytes with isolated rat CMEC. The coculture monolayer's integrity and permeability were significantly compromised by HG media exposure, which was indicated by decreased TEER without a change in tight junction protein levels in CMEC. Our study provides insight into gliovascular adaptations to increased glucose levels resulting in impaired cellular cross talk between astrocytes and CMEC, which could be one explanation for cerebral BBB disruption in diabetic conditions.

Effect of high glucose condition on glucose metabolism in primary astrocytes

In the brain, glucose enters astrocytes through glucose transporter (GLUT1) and either enters glycolysis or the glycogen shunt. Astrocytes meet the energy needs of neurons by building up and breaking down their glycogen supply. High glucose exposure causes astrocyte dysregulation, but its effects on glucose metabolism are relatively unknown. We hypothesized that high glucose conditioning induces a glycogenic state in the astrocyte, resulting in an inefficient mobilization of substrates when challenged with glucose deprivation. Using neonatal rat astrocytes, we used normal glucose (NG, 5.5 mM) vs. high glucose (HG, 25 mM) feeding media and measured cell membrane GLUT1 expression, glucose analog uptake, glycogen content, and cellular bioenergetics. This study demonstrates that HG conditioning causes increased glucose analog uptake (p < 0.05) without affecting GLUT1 membrane expression when compared to NG conditioned astrocytes. Increased glucose uptake in HG astrocytes is associated with higher baseline glycogen content compared to NG exposed astrocytes (p < 0.05). When challenged with glucose deprivation, HG astrocytes break down more than double the amount of glycogen molecules compared to NG astrocytes, although they break down a similar percentage of the starting glycogen stores (NG = 62%, HG = 55%). Additionally, HG conditioning negatively impacts astrocyte maximal respiration and glycolytic reserve capacity assessed by the Seahorse mitochondrial stress test and glycolytic stress test, respectively (p < 0.05). These results suggest that HG conditioning shifts astrocytes towards glycogen storage at baseline. Despite increased glycogen storage, HG astrocytes demonstrate decreased metabolic efficiency and capacity putting them at higher risk during extended periods of glucose deprivation.

Inhibitors of Oxidative Phosphorylation Modulate Astrocyte Inflammatory Responses through AMPK-Dependent Ptgs2 mRNA Stabilization.

Inflammatory activation of astroglia adds to the pathology of various neurological diseases. Astrocytes respond to microglia-derived cytokines such as interleukin-1α (IL-1α) with enhanced inflammatory signaling. This provokes pro-inflammatory gene expression of, among others, the eicosanoid-generating enzyme prostaglandin endoperoxide synthase 2 (Ptgs2). Whereas metabolic regulation of innate immune cell inflammatory responses is intensely studied, pathways related to how metabolism modulates inflammatory signaling in astrocytes are underexplored. Here, we examined how mitochondrial oxidative phosphorylation affects inflammatory responses towards IL-1α and tumor necrosis factor α in neonatal rat astrocytes. Blocking respiratory complex I and III or adenosine triphosphate (ATP) synthase did not affect activation of inflammatory signaling by IL-1α, but did elicit differential effects on inflammatory gene mRNA expression. Remarkably, mRNA and protein expression of Ptgs2 by IL-1α was consistently up-regulated when oxidative phosphorylation was inhibited. The increase of Ptgs2 resulted from mRNA stabilization. Mitochondrial inhibitors also increased IL-1α-triggered secretion of eicosanoids, such as prostaglandin E2, prostaglandin F2α, and 6-keto-prostaglandin F1α, as assessed by liquid chromatography/mass spectrometry. Mechanistically, attenuating oxidative phosphorylation elevated adenosine monophosphate (AMP) and activated AMP-activated protein kinase (AMPK). AMPK silencing prevented Ptgs2 up-regulation by mitochondrial inhibitors, while AMPK activators recapitulated Ptgs2 mRNA stability regulation. Our data indicate modulation of astrocyte inflammatory responses by oxidative metabolism, with relevance towards eicosanoid production.

Effects and Mechanism of Action of Neonatal Versus Adult Astrocytes on Neural Stem Cell Proliferation After Traumatic Brain Injury.

Due to the limited capacity of brain tissue to self-regenerate after traumatic brain injury (TBI), the mobilization of endogenous neural stem cells (NSCs) is a popular research topic. In the clinic, the neurogenic abilities of adults versus neonates vary greatly, which is likely related to functional differences in NSCs. Recent studies have demonstrated that the molecules secreted from astrocytes play important roles in NSC fate determination. In this study, conditioned media (CM) derived from neonatal or adult rat astrocytes, which were unstimulated or stimulated by lipopolysaccharide (LPS), were prepared to treat NSCs. Our results revealed that neonatal rat astrocytes can significantly promote the proliferation of NSCs, compared with adult rat astrocytes, regardless of whether or not they were stimulated by LPS. Furthermore, we used mass spectrometry to detect the constituents of the CM from each group. We analyzed and screened for a protein, Tenascin-C (TNC), which was highly expressed in the neonatal group but poorly expressed in the adult group. We found that TNC can bind to the NSC surface epidermal growth factor receptor and promote proliferation through the PI3K-AKT pathway in vitro. Additionally, we confirmed in vivo that TNC can promote damage repair in a rat model of TBI, through enhancing the proliferation of endogenous NSCs. We believe that these findings provide a mechanistic understanding of why neonates show better neuroregenerative abilities than adults. This also provides a potential future therapeutic target, TNC, for injury repair after TBI. Stem Cells 2019;37:1344-1356.

High glucose conditioned neonatal astrocytes results in impaired mitogenic activity in cerebral microvessel endothelial cells in co-culture

Angiogenesis is a highly complex and coordinated process in the brain. Under normal conditions, it is a vital process in growth and development, but under adverse conditions such as diabetes mellitus, it can lead to severe pathology. Astrocytes are a key constituent of the neurovascular unit and contribute to cerebral function, not only bridging the gap between metabolic supplies from blood vessels to neurons, but also regulating angiogenesis. Astrocytes affect angiogenesis by secreting angiogenic factors such as vascular endothelial growth factor (VEGF) into its microenvironment and regulating mitogenic activity in cerebral microvessel endothelial cells (CMEC). We hypothesized that astrocytes conditioned in high glucose media would produce and secrete decreased VEGF which would lead to impaired proliferation, migration, and tube formation of CMEC in vitro. Using neonatal rat astrocytes, we used normal glucose (NG, 5.5mM) vs. high glucose (HG, 25mM) feeding media and measured VEGF message and protein levels as well as secreted VEGF. We co-cultured conditioned astrocytes with isolated rat CMEC and measured mitogenic activity of endothelial cells using BrdU assay, scratch recovery assay, and tube formation assay. HG astrocytes produced and secreted decreased VEGF protein and resulted in impaired mitogenic activity when co-cultured with CMEC as demonstrated by decreased BrdU uptake, decreased scratch recovery, and slower tube formation. Our study provides insight into gliovascular adaptations to increased glucose levels resulting in impaired cellular cross-talk between astrocytes and CMEC which could be one explanation for cerebral microangiopathy seen in diabetic conditions.

RGC-32 regulates reactive astrocytosis and extracellular matrix deposition in experimental autoimmune encephalomyelitis

Extracellular matrix (ECM) deposition in active demyelinating multiple sclerosis (MS) lesions may impede axonal regeneration and can modify immune reactions. Response gene to complement (RGC)-32 plays an important role in the mediation of TGF-β downstream effects, but its role in gliosis has not been investigated. To gain more insight into the role played by RGC-32 in gliosis, we investigated its involvement in TGF-β-induced ECM expression and the upregulation of the reactive astrocyte markers α-smooth muscle actin (α-SMA) and nestin. In cultured neonatal rat astrocytes, collagens I, IV, and V, fibronectin, α-SMA, and nestin were significantly induced by TGF-β stimulation, and RGC-32 silencing resulted in a significant reduction in their expression. Using astrocytes isolated from RGC-32 knock-out (KO) mice, we found that the expression of TGF-β-induced collagens I, IV, and V, fibronectin, and α-SMA was significantly reduced in RGC-32 KO mice when compared with wild-type (WT) mice. SIS3 inhibition of Smad3 phosphorylation was also associated with a significant reduction in RGC-32 nuclear translocation and TGF-β-induced collagen I expression. In addition, during experimental autoimmune encephalomyelitis (EAE), RGC-32 KO mouse astrocytes displayed an elongated, bipolar phenotype, resembling immature astrocytes and glial progenitors whereas those from WT mice had a reactive, hypertrophied phenotype. Taken together, our data demonstrate that RGC-32 plays an important role in mediating TGF-β-induced reactive astrogliosis in EAE. Therefore, RGC-32 may represent a new target for therapeutic intervention in MS.

The Quantum Nature of Drug-Receptor Interactions: Deuteration Changes Binding Affinities for Histamine Receptor Ligands.

In this article we report a combined experimental and computational study concerning the effects of deuteration on the binding of histamine and two other histaminergic agonists to 3H-tiotidine-labeled histamine H2 receptor in neonatal rat astrocytes. Binding affinities were measured by displacing radiolabeled tiotidine from H2 receptor binding sites present on cultured neonatal rat astrocytes. Quantum-chemical calculations were performed by employing the empirical quantization of nuclear motion within a cluster model of the receptor binding site extracted from the homology model of the entire H2 receptor. Structure of H2 receptor built by homology modelling is attached in the supporting information (S1 Table) Experiments clearly demonstrate that deuteration affects the binding by increasing the affinity for histamine and reducing it for 2-methylhistamine, while basically leaving it unchanged for 4-methylhistamine. Ab initio quantum-chemical calculations on the cluster system extracted from the homology H2 model along with the implicit quantization of the acidic N–H and O–H bonds demonstrate that these changes in the binding can be rationalized by the altered strength of the hydrogen bonding upon deuteration known as the Ubbelohde effect. Our computational analysis also reveals a new mechanism of histamine binding, which underlines an important role of Tyr250 residue. The present work is, to our best knowledge, the first study of nuclear quantum effects on ligand receptor binding. The ligand H/D substitution is relevant for therapy in the context of perdeuterated and thus more stable drugs that are expected to enter therapeutic practice in the near future. Moreover, presented approach may contribute towards understanding receptor activation, while a distant goal remains in silico discrimination between agonists and antagonists based on the receptor structure.

Abstract A03: The effects of the cellular microenvironment on medulloblastoma metastasis

Abstract Medulloblastoma (MB) is the most common pediatric brain tumor, known to be highly malignant and invasive within the brain as well as extraneural locations. The mechanisms inducing MB metastasis and dissemination are not completely understood. Astrocytes, the most abundant cell type in the brain, have been shown in our lab to secrete factors that induce breast cancer brain metastasis (Wang et al., 2013). In addition to astrocytes, many brain cell types have also been implicated in the malignancy of brain tumors, including microglia, pericytes, and endothelial cells as part of the neurovascular unit. The overall goal of this study is to determine the effects of cells in the tumor microenvironment on MB metastasis. Our hypothesis is that astrocytes and/or other cell types in the neurovascular unit and microenvironment secrete factors that bring upon phenotypic changes in MB cells, leading to increased metastasis. To investigate this hypothesis, astrocyte conditioned media (ACM) from neonatal rat astrocyte cultures were used in in vitro assays to determine functional changes to Daoy MB cells. We observed that ACM (both serum free and serum depleted) in the lower chamber of a Boyden chamber Matrigel invasion assay induced invasion of Daoy cells more than 50-fold in comparison to DMEM in the lower well. Brain endothelial cell conditioned media also increased invasion of Daoy cells, which was comparable to ACM. ACM did not significantly affect proliferation or apoptosis of Daoy cells, as tested by BrdU incorporation and Caspase-3/-7 activity, respectively. When cultured appropriately in serum free ACM (+bFGF and EGF), Daoy cells were able to form larger and more proliferative neurospheres in comparison to DMEM, indicative of an increase in neural or cancer stem cell characteristics. To determine the effects of ACM on MB metastasis in vivo, we developed a zebrafish xenotransplant model. Daoy-GFP cells were injected into the hindbrain of 48 hour post fertilization Absolut FLK-mCherry zebrafish embryos. Using time-lapse confocal microscopy, we are able to image the cells and observe their movement within the microenvironment after injection. With this method we are working to determine changes in cell morphology and movement in Daoy-GFP cells that were cultured in ACM prior to injection. In order to further elucidate the mechanism in which ACM increases invasion and neurosphere formation, microarray studies are underway to pin-point a factor(s) or pathway(s) involved. Together, these data indicate that cells comprising the neurovascular unit, namely endothelial cells and astrocytes, contribute in a paracrine manner to MB metastasis and dissemination. Citation Format: Emily Gronseth, Ling Wang, Chris Koceja, Ramani Ramchandran. The effects of the cellular microenvironment on medulloblastoma metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A03.

Abstract B08: Astrocytes in the microenvironment influence breast cancer cell metastasis

Abstract Microenvironment plays a critical role in the metastasis and progression of tumor cells. Our laboratory has recently identified that cells in the brain microenvironment namely the astrocytes, play a critical role in breast cancer cell metastasis in vivo and in vitro. Previous data from our lab provide evidence that the both adult and neonatal rat astrocyte secretome influences the local matrix, thereby facilitating extravasation of cancer cells into brain parenchyma and other distant sites. In that study, we cultured and passaged breast cancer cells in the astrocyte culture media and the resultant astrocyte-conditioned breast cancer cells (ABCs) were injected via the intra-cardiac route into CB17/Severe Combined Immunodeficiency (SCID) mice. Now, we have injected the ABCs into the mammary fat pad and intravenous route, and find that irrespective of the route, these cells are highly metastatic in vivo with a few cells reaching the brain as well. ABCs also showed marked change in phenotype and became increasingly invasive, a phenotype that intriguingly was reversed when the ABCs were cultured in normal tumor media (NBCs) over subsequent passages. Based on this data, we hypothesized that solid tumors with propensity for brain metastasis respond to molecules secreted by astrocytes that induce genomic changes in cancer cells that creates a transitional state in these cells to facilitate metastasis. To investigate this hypothesis, the purpose of this study was to determine the genomic changes in ABCs, and in turn the underlying mechanisms governing ABCs metastasis. We performed microarray analysis on ABCs and NBCs and identified significant up regulation of pro-metastasis genes in ABCs with a concomitant down regulation of the same genes in NBCs thereby providing excellent correlation between genotype and phenotype. One of the genes Angiopoietin-like 4 (ANGPTL4) has been functionally implicated in breast cancer cell invasion in vitro, and knockdown of ANGPTL4 decreased migration, invasion and proliferation, as well as increased apoptosis of these cells. On a phenotypic level, we identified two different cellular morphologies in ABCs, one a small spindle like and a second large irregular cell, and FACS sorting of these two cell populations revealed that the invasive phenotype is associated with the larger irregular cell type. We also have evidence now that human astrocytes differentiated from fetal cortex also induce breast cancer cell invasion in vitro, and the ABCs invasion and metastasis phenomenon is broadly observed in other cancers including lung cancer albeit the genomic changes are not conserved suggesting a level of specificity to these changes. Collectively, these findings suggest a metastasis plan induced by astrocytes on solid tumors with a propensity to make these cells highly tumorigenic and metastatic thus providing novel avenues for intervention. Citation Format: Ling Wang, Michael Lepley, Allison Ebert, David R. Harder, Ramani Ramchandran. Astrocytes in the microenvironment influence breast cancer cell metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B08.

A Novel Dynamic Neonatal Blood-Brain Barrier on a Chip.

Studies of neonatal neural pathologies and development of appropriate therapeutics are hampered by a lack of relevant in vitro models of neonatal blood-brain barrier (BBB). To establish such a model, we have developed a novel blood-brain barrier on a chip (B3C) that comprises a tissue compartment and vascular channels placed side-by-side mimicking the three-dimensional morphology, size and flow characteristics of microvessels in vivo. Rat brain endothelial cells (RBEC) isolated from neonatal rats were seeded in the vascular channels of B3C and maintained under shear flow conditions, while neonatal rat astrocytes were cultured under static conditions in the tissue compartment of the B3C. RBEC formed continuous endothelial lining with a central lumen along the length of the vascular channels of B3C and exhibited tight junction formation, as measured by the expression of zonula occludens-1 (ZO-1). ZO-1 expression significantly increased with shear flow in the vascular channels and with the presence of astrocyte conditioned medium (ACM) or astrocytes cultured in the tissue compartment. Consistent with in vivo BBB, B3C allowed endfeet-like astrocyte-endothelial cell interactions through a porous interface that separates the tissue compartment containing cultured astrocytes from the cultured RBEC in the vascular channels. The permeability of fluorescent 40 kDa dextran from vascular channel to the tissue compartment significantly decreased when RBEC were cultured in the presence of astrocytes or ACM (from 41.0±0.9 x 10−6 cm/s to 2.9±1.0 x 10−6 cm/s or 1.1±0.4 x 10−6 cm/s, respectively). Measurement of electrical resistance in B3C further supports that the addition of ACM significantly improves the barrier function in neonatal RBEC. Moreover, B3C exhibits significantly improved barrier characteristics compared to the transwell model and B3C permeability was not significantly different from the in vivo BBB permeability in neonatal rats. In summary, we developed a first dynamic in vitro neonatal BBB on a chip (B3C) that closely mimics the in vivo microenvironment, offers the flexibility of real time analysis, and is suitable for studies of BBB function as well as screening of novel therapeutics.

Quercetin uptake into neonatal rat astrocytes

Quercetin uptake into neonatal rat astrocytes

Calcium signaling and glucose response is different in adult compared to neonatal rat astrocytes (1177.7)

Much of our mechanistic understanding of astrocytes comes from neonatal cells which may not represent the mature glial phenotype. We sought to determine if neonatal and adult astrocytes respond similarly when challenged with high glucose. Neonatal and adult rat astrocytes were cultured in 5.5 mM low glucose DMEM (LG) and then maintained in LG or switched to 25 mM high glucose DMEM (HG) for two weeks. A developmental decrease was observed in expression of the inositol trisphosphate receptor (IP3R) and the metabotropic glutamate 5 receptor (mGluR5). The L‐type calcium channel (CaV1.2) was only expressed in adult astrocytes. Adult and neonatal astrocytes both responded to HG with increased mGluR5 and decreased IP3R protein expression. Adult astrocytes also increased CaV1.2 expression following HG. Neonatal astrocytes had a greater magnitude and rate of rise of [Ca2+]i in response to ATP and glutamate compared to adult astrocytes. The [Ca2+]i responses to ATP and glutamate were similarly affected by HG, although adult astrocytes demonstrated minimal response to glutamate in all conditions. Cell viability decreased only in adult astrocytes following HG. These results suggest HG alters calcium signaling in astrocytes and developmental differences may exist in downstream pro‐survival signaling. The response of adult glia in specific disease paradigms may not be accurately reflected when using neonatal cells.Grant Funding Source: Supported by T32 HL007792, R01 HL033833, R01 HL 092105, and R01 HL105997

Regional Characteristics of Histamine Uptake into Neonatal Rat Astrocytes

Histaminergic signalling constitutes an attractive target for treatment of neuropsychiatric disorders. One obstacle to developing new pharmacological options has been failure to identify putative specific histamine transporter responsible for histamine clearance. Although high-affinity histamine uptake was detected in neonatal cortical astrocytes, its existence in other brain regions remains largely unexplored. We investigated whether cerebellar and striatal astrocytes participate in histamine clearance and evaluated the role of organic cation transporters (OCT) in astroglial histamine transport. Kinetic and pharmacological characteristics of histamine transport were determined in cultured astrocytes derived from neonatal rat cerebellum, striatum and cerebral cortex. As well as astrocytes of cortical origin, cultured striatal and cerebellar astrocytes displayed temperature-sensitive, high-affinity histamine uptake. Exposure to ouabain or Na(+)-free medium, supplemented with choline chloride markedly depressed histamine transport in cortical astrocytes. Conversely, histamine uptake in striatal and cortical astrocytes was ouabain-resistant and was only partially diminished during incubation in the absence of Na(+). Also, histamine uptake remained unaltered upon exposure to OCT inhibitor corticosterone, although OCTs were expressed in cultured astrocytes. Finally, histamine transport in cerebellar and striatal astrocytes was not sensitive to antidepressants. Despite common characteristics, cerebellar astrocytes had lower affinity, but markedly higher transport capacity for histamine compared to striatal astrocytes. Collectively, we provide evidence to suggest that cerebellar, striatal as well as cortical astrocytes possess saturable histamine uptake systems, which are not operated by OCTs. In addition, our data indicate that Na(+)-independent histamine carrier predominates in cerebellar and striatal astrocytes, whereas Na(+)-dependent transporter underlies histamine uptake in cortical astrocytes. Our findings implicate a role for histamine transporters in regulation of extracellular histamine concentration in cerebellum and striatum. Inhibition of histamine uptake might represent a viable option to modulate histaminergic neurotransmission.

Characteristics of low affinity high capacity histamine uptake into neonatal rat astrocytes

Background The neurotransmitter histamine is synthesized from histidine in histaminergic neurons. Later on it is taken up into synaptic vesicles by the vesicular monoamine transporter 2 and released into the synaptic cleft upon depolarization stimuli. The released neurotransmitter is metabolised by the enzyme histamine N-methyltransferase (HNMT) producing tele-methylhistamine (tMH). In order to be enzymatically degraded or possibly recycled, histamine must be transported either into the presynaptic neuron or into surrounding glial cells. Unlike other neurotransmitters, the mechanism and the transporters by which the histamine content within the brain is regulated is currently unresolved.

Low affinity histamine uptake into neonatal rat astrocytes does not involve OCT

Background Histamine is a double-protonated molecule with corresponding pKa values of 5.8 and 9.4. Therefore, at physiological pH, histamine exists as an equilibrium mixture of tautomeric cations: the monocation making up 96%, the dication only 3% and the rest being nonprotonated histamine. As a protonated molecule histamine most probably uses a carrier protein in order to cross the cell membrane. In the present work we wanted to determine the kinetic properties of histamine uptake and the influence of other biogenic amines on its transport.

Lipoxin A4 Inhibits 5-Lipoxygenase Translocation and Leukotrienes Biosynthesis to Exert a Neuroprotective Effect in Cerebral Ischemia/Reperfusion Injury

Lipoxin A(4) (LXA(4)), a biologically active eicosanoid with anti-inflammatory and pro-resolution properties, was recently found to have neuroprotective effects in brain ischemia. As 5-lipoxygenase (5-LOX) and leukotrienes are generally considered to aggravate cerebral ischemia/reperfusion (I/R) injury, we investigated their effects on LXA(4)-mediated neuroprotection by studying middle cerebral artery occlusion (MCAO)/reperfusion in rats and oxygen-glucose deprivation (OGD)/recovery in neonatal rat astrocyte primary cultures. LXA(4) effectively reduced infarct volumes and brain edema, and improved neurological scores in the MCAO/reperfusion experiments; this effect was partially blocked by butoxycarbonyl-Phe-Leu-Phe-Leu-Phe (Boc2), a specific antagonist of the LXA(4) receptor (ALXR). Total 5-LOX expression did not change, regardless of treatment, but LXA(4) could inhibit nuclear translocation induced by MCAO or OGD. We also found that LXA(4) inhibits the upregulation of both leukotriene B(4) (LTB(4)) and leukotriene C(4) (LTC(4)) and the phosphorylation of extracellular signal-regulated kinase (ERK) induced by MCAO or OGD. The phosphorylation of the 38-kDa protein kinase (p38) and c-Jun N-terminal kinase (JNK) was not altered throughout the experiment. These results suggest that the neuroprotective effects of LXA(4) are probably achieved by anti-inflammatory mechanisms that are partly mediated by ALXR and through an ERK signal transduction pathway.

Dopamine Stimulates Propagation ofToxoplasma gondiiTachyzoites in Human Fibroblast and Primary Neonatal Rat Astrocyte Cell Cultures

Toxoplasma gondii is an obligate intracellular parasite often found in the brain of humans. Research has shown a correlation between prevalence of antibody titers to T. gondii and psychological illness in humans. Recent studies indicate that individuals seropositive for T. gondii antibodies are more likely to develop psychotic disorders including schizophrenia, which is associated with changes in the dopamine neurotransmitter system. Dopamine in the brain may play a role in proliferation, chemoattraction, infection efficiency, or stage conversion of T. gondii . Because tachyzoites are the first developmental stage to reach the brain, the present study was conducted to determine the effects of dopamine on their development in vitro. In human fibroblast host cells, dopamine was added at either 100 nM or 250 nM to cell culture media, and the numbers of tachyzoites produced at 48 hr were determined and compared to vehicle-treated controls. An increase of tachyzoite numbers and increased destruction in cell monolayer were observed at both concentrations of dopamine. Dopamine used at 250 nM caused a significant (P < 0.05) increase in tachyzoites counts compared to controls. Dopamine antagonists (10 μM) did not significantly alter dopamine-stimulated tachyzoite production in human fibroblasts. In primary neonatal rat astrocyte cell cultures, dopamine (200 μM) significantly (P < 0.05) increased numbers of intracellular tachyzoites after 24 hr. The role that this increase plays in tachyzoite production under the stimulus of dopamine in the modulation of neural infection in humans awaits further studies.