Cultures Of Glia Research Articles

LPS and TNF alpha modulate AMPA/NMDA receptor subunit expression and induce PGE2 and glutamate release in preterm fetal ovine mixed glial cultures

BackgroundWhite matter injury (WMI) is the major antecedent of cerebral palsy in premature infants, and is often associated with maternal infection and the fetal inflammatory response. The current study explores the therapeutic potential of glutamate receptor blockade or cyclooxygenase-2 (COX-2) inhibition for inflammatory WMI.MethodsUsing fetal ovine derived mixed glia cultures exposed to tumour necrosis factor-α (TNF-α) or lipopolysaccharide (LPS), the expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and N-methyl D-aspartate (NMDA) glutamate receptors and their contribution to inflammation mediated pre-oligodendrocyte (OL) death was evaluated. The functional significance of TNF-α and COX-2 signalling in glutamate release in association with TNF-α and LPS exposure was also assessed.ResultsAMPA and NMDA receptors were expressed in primary mixed glial cultures on developing OLs, the main cell-type present in fetal white matter at a period of high risk for WMI. We show that glutamate receptor expression and configuration are regulated by TNF-α and LPS exposure, but AMPA and NMDA blockade, either alone or in combination, did not reduce pre-OL death. Furthermore, we demonstrate that glutamate and prostaglandin E2 (PGE2) release following TNF-α or LPS are mediated by a TNF-α-COX-2 dependent mechanism.ConclusionsOverall, these findings suggest that glial-localised glutamate receptors likely play a limited role in OL demise associated with chronic inflammation, but supports the COX-2 pathway as a potential therapeutic target for infection/inflammatory-mediated WMI.

Hemin uptake and release by neurons and glia

Hemin accumulates in intracerebral hematomas and may contribute to cell injury in adjacent tissue. Despite its relevance to hemorrhagic CNS insults, very little is known about hemin trafficking by neural cells. In the present study, hemin uptake and release were quantified in primary murine cortical cultures, and the effect of the hemin-binding compound deferoxamine (DFO) was assessed. Net uptake of 55Fe-hemin was similar in mixed neuron-glia, neuron, and glia cultures, but was 2.6–3.6-fold greater in microglia cultures. After washout, 40–60% of the isotope signal was released by mixed neuron-glia cultures into albumin-containing medium within 24 h. Inhibiting hemin breakdown with tin protoporphyrin IX (SnPPIX) had minimal effect, while release of the fluorescent hemin analog zinc mesoporphyrin was quantitatively similar to that of 55Fe-hemin. Isotope was released most rapidly by neurons (52.2 ± 7.2% at 2 h), compared with glia (15.6 ± 1.3%) and microglia (17.6 ± 0.54%). DFO did not alter 55Fe-hemin uptake, but significantly increased its release. Mixed cultures treated with 10 μM hemin for 24 h sustained widespread neuronal loss that was attenuated by DFO. Concomitant treatment with SnPPIX had no effect on either enhancement of isotope release by DFO or neuroprotection. These results suggest that in the presence of a physiologic albumin concentration, hemin uptake by neural cells is followed by considerable extracellular release. Enhancement of this release by DFO may contribute to its protective effect against hemin toxicity.

Amyloid β peptide-mediated neurotoxicity is attenuated by the proliferating microglia more potently than by the quiescent phenotype

BackgroundThe specific role of microglia on Aβ-mediated neurotoxicity is difficult to assign in vivo due to their complicated environment in the brain. Therefore, most of the current microglia-related studies employed the isolated microglia. However, the previous in vitro studies have suggested either beneficial or destructive function in microglia. Therefore, to investigate the phenotypes of the isolated microglia which exert activity of neuroprotective or destructive is required.ResultsThe present study investigates the phenotypes of isolated microglia on protecting neuron against Aβ-mediated neurotoxicity. Primary microglia were isolated from the mixed glia culture, and were further cultured to distinct phenotypes, designated as proliferating amoeboid microglia (PAM) and differentiated process-bearing microglia (DPM). Their inflammatory phenotypes, response to amyloid β (Aβ), and the beneficial or destructive effects on neurons were investigated. DPM may induce both direct neurotoxicity without exogenous stimulation and indirect neurotoxicity after Aβ activation. On the other hand, PAM attenuates Aβ-mediated neurotoxicity through Aβ phagocytosis and/or Aβ degradation.ConclusionsOur results suggest that the proliferating microglia, but not the differentiated microglia, protect neurons against Aβ-mediated neurotoxicity. This discovery may be helpful on the therapeutic investigation of Alzheimer’s disease.

Preferential Lentiviral Targeting of Astrocytes in the Central Nervous System

The ability to visualize and genetically manipulate specific cell populations of the central nervous system (CNS) is fundamental to a better understanding of brain functions at the cellular and molecular levels. Tools to selectively target cells of the CNS include molecular genetics, imaging, and use of transgenic animals. However, these approaches are technically challenging, time consuming, and difficult to control. Viral-mediated targeting of cells in the CNS can be highly beneficial for studying and treating neurodegenerative diseases. Yet, despite specific marking of numerous cell types in the CNS, in vivo selective targeting of astrocytes has not been optimized. In this study, preferential targeting of astrocytes in the CNS was demonstrated using engineered lentiviruses that were pseudotyped with a modified Sindbis envelope and displayed anti-GLAST IgG on their surfaces as an attachment moiety. Viral tropism for astrocytes was initially verified in vitro in primary mixed glia cultures. When injected into the brains of mice, lentiviruses that displayed GLAST IgG on their surface, exhibited preferential astrocyte targeting, compared to pseudotyped lentiviruses that did not incorporate any IgG or that expressed a control isotype IgG. Overall, this approach is highly flexible and can be exploited to selectively target astrocytes or other cell types of the CNS. As such, it can open a window to visualize and genetically manipulate astrocytes or other cells of the CNS as means of research and treatment.

Effects of borate‐based bioactive glass on neuron viability and neurite extension

Bioactive glasses have recently been shown to promote regeneration of soft tissues by positively influencing tissue remodeling during wound healing. We were interested to determine whether bioactive glasses have the potential for use in the treatment of peripheral nerve injury. In these experiments, degradable bioactive borate glass was fabricated into rods and microfibers. To study the compatibility with neurons, embryonic chick dorsal root ganglia (DRG) were cultured with different forms of bioactive borate glass. Cell viability was measured with no media exchange (static condition) or routine media exchange (transient condition). Neurite extension was measured within fibrin scaffolds with embedded glass microfibers or aligned rod sheets. Mixed cultures of neurons, glia, and fibroblasts growing in static conditions with glass rods and microfibers resulted in decreased cell viability. However, the percentage of neurons compared with all cell types increased by the end of the culture protocol compared with culture without glass. Furthermore, bioactive glass and fibrin composite scaffolds promoted neurite extension similar to that of control fibrin scaffolds, suggesting that glass does not have a significant detrimental effect on neuronal health. Aligned glass scaffolds guided neurite extension in an oriented manner. Together these findings suggest that bioactive glass can provide alignment to support directed axon growth.

Overexpression of the nuclear factor kappaB inhibitor A20 is neurotoxic after an excitotoxic injury to the immature rat brain

Background: The zinc finger protein A20 is an ubiquitinating/deubiquitinating enzyme essential for the termination of inflammatory reactions through the inhibition of nuclear factor kappaB (NF-kappaB) signaling. Moreover, it also shows anti-apoptotic activities in some cell types and proapoptotic/pronecrotic effects in others. Although it is known that the regulation of inflammatory and cell death processes are critical in proper brain functioning and that A20 mRNA is expressed in the CNS, its role in the brain under physiological and pathological conditions is still unknown. Methods: In the present study, we have evaluated the effects of A20 overexpression in mixed cortical cultures in basal conditions: the in vivo pattern of endogenous A20 expression in the control and N-methyl-d-aspartate (NMDA) excitotoxically damaged postnatal day 9 immature rat brain, and the post-injury effects of A20 overexpression in the same lesion model. Results: Our results show that overexpression of A20 in mixed cortical cultures induced significant neuronal death by decreasing neuronal cell counts by 45±9%. In vivo analysis of endogenous A20 expression showed widespread expression in gray matter, mainly in neuronal cells. However, after NMDA-induced excitotoxicity, neuronal A20 was downregulated in the neurodegenerating cortex and striatum at 10–24 hours post-lesion, and it was re-expressed at longer survival times in reactive astrocytes located mainly in the lesion border. When A20 was overexpressed in vivo 2 hours after the excitotoxic damage, the lesion volume at 3 days post-lesion showed a significant increase (20·8±7·0%). No A20-induced changes were observed in the astroglial response to injury. Conclusions: A20 is found in neuronal cells in normal conditions and is also expressed in astrocytes after brain damage, and its overexpression is neurotoxic for cortical neurons in basal mixed neuron–glia culture conditions and exacerbates postnatal brain excitotoxic damage.

Glia co-culture with neurons in microfluidic platforms promotes the formation and stabilization of synaptic contacts.

Two novel microfluidic cell culture schemes, a vertically-layered set-up and a four chamber set-up, were developed for co-culturing central nervous system (CNS) neurons and glia. The cell chambers in these devices were separated by pressure-enabled valve barriers, which permitted us to control communication between the two cell types. The unique design of these devices facilitated the co-culture of glia with neurons in close proximity (∼50-100 μm), differential transfection of neuronal populations, and dynamic visualization of neuronal interactions, such as the development of synapses. With these co-culture devices, initial synaptic contact between neurons transfected with different fluorescent markers, such as green fluorescent protein (GFP) and mCherry-synaptophysin, was imaged using high-resolution fluorescence microscopy. The presence of glial cells had a profound influence on synapses by increasing the number and stability of synaptic contacts. Interestingly, as determined by liquid chromatography-ion mobility-mass spectrometry, neuron-glia co-cultures produced elevated levels of soluble factors compared to that secreted by individual neuron or glia cultures, suggesting a potential mechanism by which neuron-glia interactions could modulate synaptic function. Collectively, these results show that communication between neurons and glia is critical for the formation and stability of synapses and point to the importance of developing neuron-glia co-culture systems such as the microfluidic platforms described in this study.

Electrical stimulation ameliorates light-induced photoreceptor degeneration in vitro via suppressing the proinflammatory effect of microglia and enhancing the neurotrophic potential of Müller cells

Many types of electrical stimulation (ES) devices have been shown to promote the survival of degenerated neural cells, such as dopaminergic neurons in the medial forebrain bundle-transected rats, ischemic-injured cortical neurons and inner-and outer-nuclear-layer cells in degenerated retina. Using a rat photic injury model, our lab previously proved the neuroprotective effect of transcorneal electrical stimulation (TCES) on apoptotic photoreceptor cells. To delineate the mechanisms that might underlie this process, the effects of ES on light-damaged photoreceptor degeneration-induced microglia and Müller cell activation were investigated in the present in vitro study. Our data showed that ES (3ms, 20Hz, 300–1600μA) increased survival among light-reared cone-derived cells (661W) cultured alongside microglia or Müller cells analyzed by LDH and TUNEL assays. The degree of rescue was found to depend on the different intensities of the ES current. The immunocytochemistry revealed that ES significantly decreased the numbers of activated microglia cells with ameboid shapes and increased the numbers of reactive gliotic Müller cells with larger soma when they were co-cultured with light-damaged 661W cells. Real-time RT-PCR and Western blotting indicated that ES which was applied to different co-cultures and 661W cell-conditioned media (661WCM)-treated glia cultures had a prominent inhibitive effect on the secretion of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in microglia and a positive regulative effect on the production of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) in Müller cells. The death rate of light-exposed 661W cells cultured with microglia was decreased significantly by the addition of neutralizing antibodies against IL-1β and TNF-α. On the other hand, the death rate of light-exposed 661W cells cultured with Müller cells was prominently increased when the co-culture was incubated in the presence of neutralizing antibody against BDNF while anti-CNTF neutralizing antibody did not induce additional exacerbation of the cell death among those 661W cells. These findings indicate the feasibility of using ES to create a nurturing environment for light-damaged photoreceptor cells. This environment is characterized by diminished microglial activation and fortified Müller cells reactive gliosis, which may have great potential in ameliorating photoreceptor damage. In this way, ES was here determined to be a novel, potent therapeutic option for delaying the progression of photoreceptor degeneration in patients suffering from retinitis pigmentosa (RP).

Appearance of neural stem cells around the damaged area following traumatic brain injury in aged rats

We have previously reported free radical production after traumatic brain injury (TBI), which induces neural stem cell (NSC) degeneration and death. However, the effects of aging on NSC proliferation around the damaged area following TBI have not been investigated. Therefore, in this study, we used 10-week (young group) and 24-month-old (aged group) rat TBI models to investigate the effects of aging on NSC proliferation around damaged tissue using immunohistochemical and ex vivo techniques. Young and aged rats received TBI. At 1, 3 and 7days after TBI, immunohistochemical and lipid peroxidation studies were performed. Immunohistochemistry revealed that the number of nestin-positive cells around the damaged area after TBI in the aged group decreased significantly when compared with those in the young group (P<0.01). However, the number of 8-hydroxy-2'-deoxyguanosine-, 4-hydroxy-2-nonenal- and single-stranded DNA (ssDNA)-positive cells and the level of peroxidation around the damaged area after TBI significantly increased in the aged group, compared with those in the young group (P<0.01). Furthermore, almost all ssDNA-positive cells in young and aged groups co-localized with NeuN and nestin staining. Ex vivo studies revealed that neurospheres, which differentiated into neurons and glia in culture, could only be isolated from injured brain tissue in young and aged groups at 3days after TBI. These results indicate that, although there were fewer NSCs that have the potential to differentiate into neurons and glia, these NSCs escaped free radical-induced degeneration around the damaged area after TBI in the aged rat brain.

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted. Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study. The principle and protocol of this methodology have been described in the literature. Additionally, alternate methodologies to isolate primary microglia are well described. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures. It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

(−)-Epigallocatechin-3-gallate increases the number of neural stem cells around the damaged area after rat traumatic brain injury

A major component of green tea is (-)-epigallocatechin gallate (EGCG), which has strong antioxidant properties. Here, we investigated the effect of EGCG on neural stem cell (NSC) proliferation around the damaged area following traumatic brain injury (TBI). In this study, male Wistar rats that had access to normal drinking water, or water containing 0.1% (w/v) EGCG, ad libitum received TBI at 10weeks of age. Immunohistochemistry revealed that the number of nestin-positive cells around the damaged area after TBI in the EGCG treatment group increased significantly compared with the normal water group (P<0.05). However, the number of 8-hydroxy-2'-deoxyguanosine-, 4-hydroxy-2-nonenal-, single-stranded DNA (ssDNA)-positive cells and the level of peroxidation around the damaged area after TBI significantly decreased in the EGCG treatment group when compared with the water group (P<0.05). Furthermore, in contrast to the EGCG group, almost all ssDNA-positive cells in the water group co-localized with NeuN and nestin-staining. Ex vivo studies revealed that spheres could only be isolated from injured brain tissue in the water group at 3days following TBI. However, in the EGCG group, spheres could be isolated at both 3 and 7days following TBI. A greater number of spheres could be isolated from the EGCG group, which differentiated into neurons and glia in culture without basic fibroblast growth factor. These results indicate that consumption of water containing EGCG pre- and post-TBI inhibits free radical-induced degradation of NSCs, which have the potential to differentiate into neurons and glia around the area of damage following TBI.

Age-changes in gene expression in primary mixed glia cultures from young vs. old rat cerebral cortex are modified by interactions with neurons

Astrocytic GFAP expression increases during normal aging in many brain regions and in primary astrocyte cultures derived from aging rodent brains. As shown below, we unexpectedly found that the age-related increase of GFAP expression was suppressed in mixed glia (astrocytes+microglia). However, the age-related increase of GFAP was observed when E18 neurons were co-cultured with mixed glia. Thus, the presence of microglia can suppress the age-related increase of GFAP, in primary cultures of astrocytes. To more broadly characterize how aging and co-culture with neurons alters glial gene expression, we profiled gene expression in mixed glia from young (3 mo) and old (24 mo) male rat cerebral cortex by Affymetrix microarray (Rat230 2.0). The majority of age changes were independent of the presence of neurons. Overall, the expression of twofold more genes increased with age than decreased with age. The minority of age changes that were either suppressed or revealed by the presence of neurons may be useful to analyze glial-neuron interaction during aging. Some in vitro changes are shared with those of aging rat hippocampus in studies from the Landfield group (Rowe et al., 2007; Kadish et al., 2009).

Enteric glia are multipotent in culture but primarily form glia in the adult rodent gut

It is unclear whether neurogenesis occurs in the adult mammalian enteric nervous system (ENS). Neural crest-derived cells capable of forming multilineage colonies in culture, and neurons and glia upon transplantation into chick embryos, persist throughout adult life in the mammalian ENS. In this study we sought to determine the physiological function of these cells. We discovered that these cells could be identified based on CD49b expression and that they had characteristics of enteric glia, including p75, GFAP, S100B, and SOX10 expression. To test whether new neurons or glia arise in the adult gut under physiological conditions, we marked dividing progenitors with a thymidine analog in rodents under steady-state conditions, or during aging, pregnancy, dietary changes, hyperglycemia, or exercise. We also tested gut injuries including inflammation, irradiation, benzalkonium chloride treatment, partial gut stenosis, and glial ablation. We readily observed neurogenesis in a neurogenic region of the central nervous system, but not reproducibly in the adult ENS. Lineage tracing of glial cells with GFAP-Cre and GFAP-CreERT2 also detected little or no adult ENS neurogenesis. Neurogenesis in the adult gut is therefore very limited under the conditions we studied. In contrast, ENS gliogenesis was readily observed under steady-state conditions and after injury. Adult enteric glia thus have the potential to form neurons and glia in culture but are fated to form mainly glia under physiological conditions and after the injuries we studied.

Inhibition of Microglial Phagocytosis Is Sufficient To Prevent Inflammatory Neuronal Death

It is well-known that dead and dying neurons are quickly removed through phagocytosis by the brain's macrophages, the microglia. Therefore, neuronal loss during brain inflammation has always been assumed to be due to phagocytosis of neurons subsequent to their apoptotic or necrotic death. However, we report in this article that under inflammatory conditions in primary rat cultures of neurons and glia, phagocytosis actively induces neuronal death. Specifically, two inflammatory bacterial ligands, lipoteichoic acid or LPS (agonists of glial TLR2 and TLR4, respectively), stimulated microglial proliferation, phagocytic activity, and engulfment of ∼30% of neurons within 3 d. Phagocytosis of neurons was dependent on the microglial release of soluble mediators (and peroxynitrite in particular), which induced neuronal exposure of the eat-me signal phosphatidylserine (PS). Surprisingly, however, eat-me signaling was reversible, so that blocking any step in a phagocytic pathway consisting of PS exposure, the PS-binding protein milk fat globule epidermal growth factor-8, and its microglial vitronectin receptor was sufficient to rescue up to 90% of neurons without reducing inflammation. Hence, our data indicate a novel form of inflammatory neurodegeneration, where inflammation can cause eat-me signal exposure by otherwise viable neurons, leading to their death through phagocytosis. Thus, blocking phagocytosis may prevent some forms of inflammatory neurodegeneration, and therefore might be beneficial during brain infection, trauma, ischemia, neurodegeneration, and aging.

Glutamatergic Neurons in Rodent Models Respond to Nanoscale Particulate Urban Air Pollutants in Vivo and in Vitro

Background: Inhalation of airborne particulate matter (PM) derived from urban traffic is associated with pathology in the arteries, heart, and lung; effects on brain are also indicated but are less documented.Objective: We evaluated rodent brain responses to urban nanoscale (< 200 nm) PM (nPM).Methods: Ambient nPM collected near an urban freeway was transferred to aqueous suspension and reaerosolized for 10-week inhalation exposure of mice or directly applied to rat brain cell cultures.Results: Free radicals were detected by electron paramagnetic resonance in the nPM 30 days after initial collection. Chronic inhalation of reaerosolized nPM altered selected neuronal and glial activities in mice. The neuronal glutamate receptor subunit (GluA1) was decreased in hippocampus, whereas glia were activated and inflammatory cytokines were induced [interleukin-1α (IL-1α), tumor necrosis factor-α (TNFα)] in cerebral cortex. Two in vitro models showed effects of nPM suspensions within 24–48 hr of exposure that involved glutamatergic functions. In hippocampal slice cultures, nPM increased the neurotoxicity of NMDA (N-methyl-d-aspartic acid), a glutamatergic agonist, which was in turn blocked by the NMDA antagonist AP5 [(2R)-amino-5-phosphonopentanoate]. In embryonic neuron cultures, nPM impaired neurite outgrowth, also blocked by AP5. Induction of IL-1α and TNFα in mixed glia cultures required higher nPM concentrations than did neuronal effects. Because conditioned media from nPM-exposed glia also impaired outgrowth of embryonic neurites, nPM can act indirectly, as well as directly, on neurons in vitro.Conclusions: nPM can affect embryonic and adult neurons through glutamatergic mechanisms. The interactions of nPM with glutamatergic neuronal functions suggest that cerebral ischemia, which involves glutamatergic excitotoxicity, could be exacerbated by nPM.

Amyloid-β Protein Oligomer at Low Nanomolar Concentrations Activates Microglia and Induces Microglial Neurotoxicity

Amyloid-β Protein Oligomer at Low Nanomolar Concentrations Activates Microglia and Induces Microglial Neurotoxicity

HMGB1 Acts on Microglia Mac1 to Mediate Chronic Neuroinflammation That Drives Progressive Neurodegeneration

What drives the gradual degeneration of dopamine neurons in Parkinson's disease (PD), the second most common neurodegenerative disease, remains elusive. Here, we demonstrated, for the first time, that persistent neuroinflammation was indispensible for such a neurodegenerative process. 1-Methyl-4-phenylpyridinium, lipopolysaccharide (LPS), and rotenone, three toxins often used to create PD models, produced acute but nonprogressive neurotoxicity in neuron-enriched cultures. In the presence of microglia (brain immune cells), these toxins induced progressive dopaminergic neurodegeneration. More importantly, such neurodegeneration was prevented by removing activated microglia. Collectively, chronic neuroinflammation may be a driving force of progressive dopaminergic neurodegeneration. Conversely, ongoing neurodegeneration sustained microglial activation. Microglial activation persisted only in the presence of neuronal damage in LPS-treated neuron-glia cultures but not in LPS-treated mixed-glia cultures. Thus, activated microglia and damaged neurons formed a vicious cycle mediating chronic, progressive neurodegeneration. Mechanistic studies indicated that HMGB1 (high-mobility group box 1), released from inflamed microglia and/or degenerating neurons, bound to microglial Mac1 (macrophage antigen complex 1) and activated nuclear factor-κB pathway and NADPH oxidase to stimulate production of multiple inflammatory and neurotoxic factors. The treatment of microglia with HMGB1 led to membrane translocation of p47(phox) (a cytosolic subunit of NADPH oxidase) and consequent superoxide release, which required the presence of Mac1. Neutralization of HMGB1 and genetic ablation of Mac1 and gp91(phox) (the catalytic submit of NADPH oxidase) blocked the progressive neurodegeneration. Our findings indicated that HMGB1-Mac1-NADPH oxidase signaling axis bridged chronic neuroinflammation and progressive dopaminergic neurodegeneration, thus identifying a mechanistic basis for chronic PD progression.

Epigenetic regulation of the calcitonin gene–related peptide gene in trigeminal glia

The neuropeptide calcitonin gene-related peptide (CGRP) plays a key role in migraine. CGRP gene expression involves an enhancer that is active in neurons, yet inactive in glia. In this report, we analyze epigenetic modifications that allow enhancer activation in glia. DNA methylation and histone acetylation states were measured in rat and human- model cell lines and primary cultures of rat trigeminal ganglia glia. The functional consequence of altering the chromatin state was determined by quantitative measurements of both calcitonin (CT) and CGRP mRNAs. A hypermethylated CpG island flanking the enhancer was identified in glia and non-expressing cell lines. In addition, the chromatin was hypoacetylated. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine induced CT mRNA ~30-fold in glial cultures. Treatment with a histone deacetylase inhibitor alone had little effect; however, the combination of inhibitors yielded a synergistic ~80-fold increase in CT and ~threefold increase in CGRP mRNA. Treated glia contained CT precursor (pro-CT) immunoreactivity. Epigenetic modulation is sufficient to induce the CGRP gene in glia. Because the CGRP gene is systemically activated by inflammatory conditions, this suggests that glial pro-CT may be an unexplored biomarker during migraine.

Engineered Neuronal Circuits: A New Platform for Studying the Role of Modular Topology

Neuron–glia cultures serve as a valuable model system for exploring the bio-molecular activity of single cells. Since neurons in culture can be conveniently recorded with great fidelity from many sites simultaneously, it has long been suggested that uniform cultured neurons may also be used to investigate network-level mechanisms pertinent to information processing, activity propagation, memory, and learning. But how much of the functionality of neural circuits can be retained in vitro remains an open question. Recent studies utilizing patterned networks suggest that they provide a most useful platform to address fundamental questions in neuroscience. Here we review recent efforts in the realm of patterned networks’ activity investigations. We give a brief overview of the patterning methods and experimental approaches commonly employed in the field, and summarize the main results reported in the literature. The general picture that emerges from these reports indicates that patterned networks with uniform connectivity do not exhibit unique activity patterns. Rather, their activity is very similar to that of unpatterned uniform networks. However, by breaking the connectivity homogeneity, using a modular architecture, it is possible to introduce pronounced topology-related gating and delay effects. These findings suggest that patterned cultured networks may serve as a new platform for studying the role of modularity in neuronal circuits.

Release of ATP from avian Müller glia cells in culture

ATP can be released from neurons and act as a neuromodulator in the nervous system. Besides neurons, cortical astrocytes also are capable of releasing ATP from acidic vesicles in a Ca2+-dependent way. In the present work, we investigated the release of ATP from Müller glia cells of the chick embryo retina by examining quinacrine staining and by measuring the extracellular levels of ATP in purified Müller glia cultures. Our data revealed that glial cells could be labeled with quinacrine, a reaction that was prevented by incubation of the cells with 1μM bafilomycin A1 or 2μM Evans blue, potent inhibitors of vacuolar ATPases and of the vesicular nucleotide transporter, respectively. Either 50mM KCl or 1mM glutamate was able to decrease quinacrine staining of the cells, as well as to increase the levels of ATP in the extracellular medium by 77% and 89.5%, respectively, after a 5min incubation of the cells. Glutamate-induced rise in extracellular ATP could be mimicked by 100μM kainate (81.5%) but not by 100μM NMDA in medium without MgCl2 but with 2mM glycine. However, both glutamate- and kainate-induced increase in extracellular ATP levels were blocked by 50μM of the glutamatergic antagonists DNQX and MK-801, suggesting the involvement of both NMDA and non-NMDA receptors. Extracellular ATP accumulation induced by glutamate was also blocked by incubation of the cells with 30μM BAPTA-AM or 1μM bafilomycin A1. These results suggest that glutamate, through activation of both NMDA and non-NMDA receptors, induces the release of ATP from retinal Müller cells through a calcium-dependent exocytotic mechanism.