Reactive oxygen species modulate the differentiation of neurons in clonal cortical cultures

Reactive oxygen species (ROS) are important regulators of intracellular signaling. We examined the expression of ROS during rat brain development and explored their role in differentiation using cortical cultures. High levels of ROS were found in newborn neurons. Neurons produced ROS, not connected with cell death, throughout embryogenesis and postnatal stages. By P20, ROS-producing cells were found only in neurogenic regions. Cells with low levels of ROS, isolated from E15 brains by FACS, differentiated into neurons, oligodendrocytes, and astrocytes in clonal cultures. Neurons produced high ROS early in culture and later differentiated into two types: large pyramidal-like neurons that fired no or only a single action potential and smaller neurons that expressed nuclear calretinin and fired repeated action potentials. Antioxidant treatment did not alter neuron number but increased the ratio of small to large neurons. These findings suggest that modulation of ROS levels influences multiple aspects of neuronal differentiation.     

Reactive oxygen species modulate neuronal excitability in rat intrinsic cardiac ganglia

Reactive oxygen species (ROS) are produced as by-products of oxidative metabolism and occur in the heart during ischemia and coronary artery reperfusion. The effects of ROS on the electrophysiological properties of intracardiac neurons were investigated in the intracardiac ganglion (ICG) plexus in situ and in dissociated neurons from neonatal and adult rat hearts using the whole-cell patch clamp recording configuration. Bath application of ROS donors, hydrogen peroxide (H₂O₂) and tert-butyl hydroperoxide (t-BHP) hyperpolarized, and increased the action potential duration of both neonatal and adult ICG neurons. This action was also recorded in ICG neurons in an adult in situ ganglion preparation. H₂O₂ and t-BHP also inhibited voltage-gated calcium channel (VGCC) currents and shifted the current–voltage (I–V) relationship to more hyperpolarized potentials. In contrast, H₂O₂ increased the amplitude of the delayed rectifier K⁺ current in neonatal ICG neurons. In neonatal ICG neurons, bath application of either superoxide dismutase (SOD) or catalase, scavengers of ROS, prior to H₂O₂ attenuated the hyperpolarizing shift but not the inhibition of VGCC by H₂O₂. In contrast, in adult ICG neurons, application of SOD alone had no effect upon either VGCC current amplitude or the I–V relationship, whereas application of SOD prior to H₂O₂ exposure abolished both the H₂O₂-mediated hyperpolarizing shift and inhibition. These data indicate that ROS alter depolarization-activated Ca²⁺ and K⁺ conductances which underlie neuronal excitability of ICG neurons. This affects action potential duration and therefore probably modifies autonomic control of the heart during ischemia/reperfusion.     

Secreted factors from ventral telencephalon induce the differentiation of GABAergic neurons in cortical cultures

It is widely believed that the pyramidal cells and interneurons of the cerebral cortex are distinct in their origin, lineage and genetic make up. In view of these findings, the current thesis is that the phenotype determination of cortical neurons is primarily directed by genetic mechanisms. Using in vitro assays, the present study demonstrates that secreted factors from ganglionic eminence (GE) of the ventral telencephalon have the potency to induce the differentiation of a subset of cortical neurons towards gamma-aminobutyric acid (GABA)ergic lineage. Characterization of cortical cultures that were exposed to medium derived from GE illustrated a significant increase in the number of GABA-, calretinin- and calbindin-positive neurons. Calcium imaging together with pharmacological studies showed that the application of exogenous medium significantly elevated the intracellular calcium transients in cortical neurons through the activation of ionotropic glutamate receptors. The increase in GABA+ neurons appeared to be associated with the elevated calcium activity; treatment with blockers specific for glutamate receptors abolished both the synchronized transients and reduced the differentiation of GABAergic neurons. Such studies demonstrate that although intrinsic mechanisms determine the fate of cortical interneurons, extrinsic factors have the potency to influence their neurochemical differentiation and contribute towards their molecular diversity.     

Reactive oxygen species are involved in BMP-induced dendritic growth in cultured rat sympathetic neurons

Previous studies have shown that bone morphogenetic proteins (BMPs) promote dendritic growth in sympathetic neurons; however, the downstream signaling molecules that mediate the dendrite promoting activity of BMPs are not well characterized. Here we test the hypothesis that reactive oxygen species (ROS)-mediated signaling links BMP receptor activation to dendritic growth. In cultured rat sympathetic neurons, exposure to any of the three mechanistically distinct antioxidants, diphenylene iodinium (DPI), nordihydroguaiaretic acid (NGA) or desferroxamine (DFO), blocked de novo BMP-induced dendritic growth. Addition of DPI to cultures previously induced with BMP to extend dendrites caused dendritic retraction while DFO and NGA prevented further growth of dendrites. The inhibition of the dendrite promoting activity of BMPs by antioxidants was concentration-dependent and occurred without altering axonal growth or neuronal cell survival. Antioxidant treatment did not block BMP activation of SMAD 1,5 as determined by nuclear localization of these SMADs. While BMP treatment did not cause a detectable increase in intracellular ROS in cultured sympathetic neurons as assessed using fluorescent indicator dyes, BMP treatment increased the oxygen consumption rate in cultured sympathetic neurons as determined using the Seahorse XF24 Analyzer, suggesting increased mitochondrial activity. In addition, BMPs upregulated expression of NADPH oxidase 2 (NOX2) and either pharmacological inhibition or siRNA knockdown of NOX2 significantly decreased BMP-7 induced dendritic growth. Collectively, these data support the hypothesis that ROS are involved in the downstream signaling events that mediate BMP7-induced dendritic growth in sympathetic neurons, and suggest that ROS-mediated signaling positively modulates dendritic complexity in peripheral neurons.     

Birth-date-dependent segregation of the mouse cerebral cortical neurons in reaggregation cultures

Cerebral cortical neurons form a six-layered structure in which their position depends on their birth date. This developmental process requires the presence of Reelin, which is secreted by Cajal-Retzius cells in the cortical marginal zone (MZ). However, it is still unclear whether the migration from the ventricular zone (VZ) to beneath the MZ is essential for the neurons to segregate into layers. Previous transplantation studies of ferret cerebral cortical neurons suggested that their ultimate laminar fate is, at least to some extent, determined in the VZ but it is unknown how 'laminar fate' eventually positions cells in a specific layer. To explore the segregation properties of mouse cortical cells that have not yet arrived beneath the MZ, embryonic day (E)16 VZ and intermediate zone (IMZ) cells were dissociated and allowed to reaggregate for 1-4 days in vitro. The results suggested that the migrating neurons in the IMZ at E16 preferentially located near the centre of the aggregates, more than did the proliferative cells from the VZ. The birth-date labelling followed by the dissociation-reaggregation culture suggested that the segregation properties of the E16 IMZ was characteristic of the E14-born cells, which were migrating in the IMZ at E16, but they were not general properties of migrating IMZ cells. This birth-date-dependent segregation mechanism was also observed in the Reelin signalling-deficient yotari cells. These findings suggest that cortical neurons acquire a birth-date-dependent segregation mechanism before their somas reach the MZ.     

Reactive oxygen species initiate a metabolic collapse in hippocampal slices: potential trigger of cortical spreading depression

Excessive accumulation of reactive oxygen species (ROS) underlies oxidative damage. We find that in hippocampal slices, decreased activity of glucose-based antioxidant system induces a massive, abrupt, and detrimental change in cellular functions. We call this phenomenon metabolic collapse (MC). This collapse manifested in long-lasting silencing of synaptic transmission, abnormal oxidation of NAD(P)H and FADH₂ associated with immense oxygen consumption, and massive neuronal depolarization. MC occurred without any preceding deficiency in neuronal energy supply or disturbances of ionic homeostasis and spread throughout the hippocampus. It was associated with a preceding accumulation of ROS and was largely prevented by application of an efficient antioxidant Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl). The consequences of MC resemble cortical spreading depression (CSD), a wave of neuronal depolarization that occurs in migraine, brain trauma, and stroke, the cellular initiation mechanisms of which are poorly understood. We suggest that ROS accumulation might also be the primary trigger of CSD. Indeed, we found that Tempol strongly reduced occurrence of CSD in vivo, suggesting that ROS accumulation may be a key mechanism of CSD initiation.     

Reactive oxygen and nitrogen species in Alzheimer's disease

Age- related neurodegenerative diseases, especially Alzheimer's disease (AD), are an important health problem globally. AD is clinically characterized by loss of memory, reasoning and speech. The frequency of the disease reaches to 20-40% in the population over the age of 85. Autopsy findings have indicated the presence of senile plaques and neurofibrillary tangles in the brains of patients with AD. These two lesions can be seen in small numbers during normal aging of the brain but occur in large amounts during AD. Although the initiating causes leading to AD are unknown, oxidative damage appears to play an important role in the slowly progressive neuronal death that is characteristic of AD. Indeed, in addition to the presence of senile plaques and neurofibrillary tangles, postmortem analysis of AD brain has also identified markers of oxidative stress including protein nitrotyrosine, carbonyls in proteins, lipid oxidation products and oxidized DNA bases. This review discusses the role of reactive oxygen and nitrogen species in the pathogenesis of AD and examines the relevance of antioxidant therapy in altering and/or inhibiting neurodegeneration associated with the disease.     

Reactive oxygen species in NMDA receptor-mediated glutamate neurotoxicity

In search of endogenous protective substances that inhibit neurotoxic action of glutamate and nitric oxide (NO), we found that brain-derived neurotrophic factor (BDNF), acting on TrkB receptor tyrosine kinase, inhibited neurotoxicity induced by glutamate and NO donors in cultured cortical neurons. In co-cultures of the mesencephalon and striatum, projection of mesencephalic dopamine neurons to the striatum attenuated N-methyl-d-aspartate (NMDA)-induced cytotoxicity in dopamine neurons themselves. Growth factors such as neurotrophins, which the target cells in the striatum would synthesize and secrete, may offer the protection of dopamine neurons against glutamate neurotoxicity.     

Neuropeptide Y immunoreactive neurons in murine trisomy 16 cortical cultures

Neuropeptide Y (NPY)-containing neurons are depleted in the cortices of individuals with Alzheimer disease (AD), yet spared in the striatum of patients with Huntington chorea. It is unknown whether this neuronal phenotype is inherently susceptible to the neurodegenerative processes that are a hallmark of AD. To study this question, the murine trisomy 16 model of Down syndrome and Alzheimer disease was investigated. Since trisomic fetuses diein utero, studies were carried out on primary cultures of dissociated cortical neurons. These were prepared from 15-d gestational trisomy 16 fetuses and their littermate euploid controls, and examined by immunocytochemical staining for neuropeptide Y at 7 and 12 d in vitro. Trisomy 16 neurons were also grown on euploid glial carpets, whereas euploid neurons were grown on trisomic glia. The results demonstrate a significant increase in the number of NPY neurons and a stunting in the dendritic arbor of these neurons in trisomic vs euploid cortex. Both of these parameters could be normalized by direct contact with euploid glia. When euploid cortex was plated on trisomic glia, the number of NPY neurons and their morphology were altered so that they began to resemble trisomic NPY cortical neurons. These results indicate a dysregulation of NPY neuronal expression and differentiation in trisomy 16 cortex that are modifiable by interaction with euploid glia and imply an abnormal trophic (glial) environment in trisomic cortex.     

PSA-NCAM modulates BDNF-dependent survival and differentiation of cortical neurons

We show that the loss or inactivation of the polysialic acid (PSA) tail of neural cell adhesion molecule (NCAM) on rat cortical neurons in culture leads to reduced differentiation and survival. The mechanism by which this negative effect is mediated appears to involve the neuronal response to brain-derived neurotrophic factor (BDNF): (i) in the absence of PSA or in the presence of excess free PSA added to the culture medium, BDNF-induced cell signalling is reduced; (ii) the addition of exogenous BDNF to the medium reverses the effect of PSA loss or inactivation. These data suggest that PSA-NCAM, previously shown to modulate cell migration and plasticity, is needed for an adequate sensitivity of neurons to BDNF.     

Medullary norepinephrine neurons modulate local oxygen concentrations in the bed nucleus of the stria terminalis

Neurovascular coupling is understood to be the underlying mechanism of functional hyperemia, but the actions of the neurotransmitters involved are not well characterized. Here we investigate the local role of the neurotransmitter norepinephrine in the ventral bed nucleus of the stria terminalis (vBNST) of the anesthetized rat by measuring O₂, which is delivered during functional hyperemia. Extracellular changes in norepinephrine and O₂ were simultaneously monitored using fast-scan cyclic voltammetry. Introduction of norepinephrine by electrical stimulation of the ventral noradrenergic bundle or by iontophoretic ejection induced an initial increase in O₂ levels followed by a brief dip below baseline. Supporting the role of a hyperemic response, the O₂ increases were absent in a brain slice containing the vBNST. Administration of selective pharmacological agents demonstrated that both phases of this response involve β-adrenoceptor activation, where the delayed decrease in O₂ is sensitive to both α- and β-receptor subtypes. Selective lesioning of the locus coeruleus with the neurotoxin DSP-4 confirmed that these responses are caused by the noradrenergic cells originating in the nucleus of the solitary tract and A1 cell groups. Overall, these results support that non-coerulean norepinephrine release can mediate activity-induced O₂ influx in a deep brain region.     

Identification of GABA neurons in rat cortical cultures by GABA uptake autoradiography

Autoradiographic studies of rat cortical cultures were conducted with tritiated transmitters and related drugs. Autoradiographs prepared from cultures incubated in [³H]GABA showed selective labeling: dense accumulations of silver grains over the somas and all processes of approximately 30–50% of the neuronal population, few grains over the non-neuronal cells. This labeling was blocked by diaminobutyric acid (DABA) and sodium-free media but not by β-alanine and thus has the characteristics of GABA uptake in other neuronal systems. There were no obvious differences in the size, shape, number of processes or distribution in the culture between neurons which accumulated GABA and those which did not. Similar cultures incubated in either [³H]glycine or [³H]glutamate and processed for autoradiography resulted in a much different distribution of silver grains than that seen for [³H]GABA. Following incubation in [³H]glycine, silver grains were distributed uniformly over all cells in the culture, both neuronal and non-neuronal. This distribution suggests a metabolic and not a neurotransmitter role for glycine in the cultures, as would be expected of neuronal cells derived from cerebral cortex. Glutamate incubations resulted in the appearance of silver grains over only the non-neuronal cells with very few over the neuronal population. Autoradiograms were also prepared following incubation in the potent GABA receptor agonist [³H]muscimol. These autoradiograms were indistinguishable from those obtained following [³H]GABA incubation. Thus, a finite population of neurons was densely labeled, the labeling was blocked by the GABA uptake inhibitors DABA, nipecotic acid, guvacine and Na⁺-free media, while substances which interact with the GABA receptor, bicuculline methiodide, THIP, isoguvacine and the noncompetitive antagonist, picrotoxin, were without effect. These results demonstrate that the affinity of muscimol for the GABA uptake site far outweighs its affinity for the GABA receptor site in autoradiographic experiments where intact cells are employed, presumably because its binding to receptors is fleeting. Therefore, muscimol autoradiography may not be informative about GABA receptor localization.These autoradiographic studies suggest that nearly half the neurons in our culture system are GABA neurons but disclosed no morphological handle for GABA neurons.     

自由基介导兴奋性氨基酸对培养皮层神经细胞毒性的研究

本文在体外对新生大鼠（０～１ｄ）大脑皮层神经细胞进行原代培养的基础上，建立谷氨酸（Ｇｌｕ）对培养的皮层神经细胞兴奋毒损伤模型。通过检测乳酸脱氢酶释出率和形态学观察，证实了Ｇｌｕ对神经细胞存在兴奋毒作用。通过测定不同条件下Ｇｌｕ兴奋毒对培养的神经细胞造成损伤时细胞膜脂质过氧化物（ＬＰＯ）浓度，发现膜ＬＰＯ浓度与Ｇｌｕ剂量及作用时间紧密相关，表明Ｇｌｕ对神经细胞兴奋毒作用有自由基产生并介导了神经细胞的损伤。     

Ascorbate-induced differentiation of embryonic cortical precursors into neurons and astrocytes

A specific role for ascorbate (AA) in brain development has been postulated based on a rise of AA levels in fetal brain (Kratzing et al., 1985). To evaluate the role of AA during CNS development, we analyzed the survival, proliferation, and differentiation of AA-treated CNS precursor cells isolated from rat embryonic cortex. Immunocytochemical analyses revealed that AA promoted the in vitro differentiation of CNS precursor cells into neurons and astrocytes in a cell density-dependent manner. Additionally, AA increased the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) of postmitotic neurons in primary neuronal cultures. Differential expression analysis of genes specific to neuronal or glial differentiation revealed an AA-dependent increase in the expression of genes that could potentially compound the effects of AA on cell differentiation. These data suggest that AA may act in the developing brain to stimulate the generation of CNS neurons and glia, thereby assisting in the formation of neural circuits by promoting the acquisition of neuronal synaptic functions.     

Modulation of phospholipase A2 activity in primary cultures of rat cortical neurons

Summary. In neurons, phospholipase A₂ (PLA₂) plays a central role in the regulation of membrane phospholipid metabolism. We have addressed the pharmacological modulation of PLA₂ in primary cultures of rat cortical neurons. Inhibition curves were obtained in 4 day-in-culture neurons treated for 30 minutes with either the dual PLA₂ inhibitor methyl arachidonyl fluorophosphonate (MAFP), or the iPLA₂ inhibitor bromoenol lactone (BEL). Full inhibition was achieved with 100 and 250 μM of MAFP, or 10 and 20 μM of BEL. Conversely, a dose-dependent activation of PLA₂ was obtained with 10–20 μg/ml of melitin. PLA₂ inhibition with MAFP or BEL was not acutely toxic for cultured neurons. However, sustained inhibition of the enzyme precluded the development of neurites, and resulted in long-term loss of neuronal viability. We present a model of pharmacological challenge of PLA₂ in vitro, which can be further used to address the involvement of the enzyme in neurodevelopment and neurodegeneration models.     

Newborn neurons acquire high levels of reactive oxygen species and increased mitochondrial proteins upon differentiation from progenitors

A population of embryonic rat cortical cells cultured in the presence of FGF2 and having neuronal morphology expressed higher levels of reactive oxygen species (ROS) than did progenitor cells, astrocytes, and several cell lines of neuronal and non-neuronal origin. ROS were assessed using 5-(and-6)-chlormethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCF-DA), and high levels persisted in the presence of antioxidants or lowered levels of ambient oxygen. Greater than 95% of high ROS-producing cells, isolated by fluorescence-activated cell sorting, expressed the neuronal marker beta III tubulin. These cells did not incorporate BrdU or express nestin, unlike low ROS-producing cells, 99% of which exhibited both of these characteristics. Upon growth factor removal, low ROS-expressing cells differentiated into neurons and astrocytes and these neurons expressed high levels of ROS, indicating that ROS accumulation accompanies the differentiation of progenitors into neurons. ROS levels were decreased by added superoxide dismutase and catalase, suggesting that both superoxide and hydrogen peroxide contribute to the ROS signal. High ROS-expressing cells also contained higher levels of several mitochondrial respiratory chain components. Although ROS have been associated with conditions that lead to cell death, our results and recent studies on the role of ROS as regulators of signal pathways are consistent with the possibility that ROS play a role in the development of the neuronal phenotype. Moreover, the differential production of ROS provides a useful method to isolate from mixed populations cells that are highly enriched for either progenitor cells or neurons.     

Morphological differentiation of neuropeptide Y neurons in aggregate cultures of dissociated fetal cortical cells: a model system for glia-neuron paracrine interactions

The temporal changes in the morphological profiles of neuropeptide Y (NPY) neurons and their topographical relationship with glial cells (astrocytes) were characterized in aggregate cultures derived from fetal cortical tissue using immunocytochemical procedures. On day 6 of culture, structures labelled with NPY antibodies were small and uneven in size but many resembled neuronal cell bodies. On day 14, neuronal perikarya were well defined and several morphological types of NPY neurons could be distinguished most of which gave rise to beaded processes: unipolar or multipolar bitufted neurons whose processes branch in close proximity to the cell body; bipolar neurons; and multipolar neurons. On day 23, heavily punctate and asymmetrically labelled cell bodies were dispersed throughout the aggregate; neuronal processes were less conspicuous. At 14 and 23 days, cells expressing glial fibrillary acidic protein (GFAP) and neuronal specific enolase (NSE) were abundantly distributed throughout the aggregate. Using a double immunoreaction on 14-day-old aggregates revealed that GFAP + cells and their processes were in close apposition to and engulfing the NPY neurons. Thus, dissociated fetal NPY neurons undergo morphological differentiation in culture along with astrocytes (GFAP +) and other neuronal cell types (NSE +). Based on the topographical association of astrocytes and neurons, particularly NPY neurons, we propose that the aggregate culture system can serve as a model to study the role of paracrine interactions in the regulation of the expression of NPY.     

Morphological and physiological differentiation of Purkinje neurons in cultures of rat cerebellum

During ontogeny, vertebrate CNS neurons differentiate from relatively simple stem cells to complex units that express unique morphological and electrophysiological characteristics. We have examined several aspects of this developmental process in an identified CNS neuronal type, the Purkinje neuron (PN) of the cerebellum. Our approach has included the use of a tissue culture preparation and immunohistochemical and electrophysiological techniques. Using immunohistochemical techniques, we have identified immature PNs in culture and examined their morphological and synaptic development. These studies have shown that PNs undergo extensive morphological and synaptic development in culture, the morphological characteristics of the immature PNs in culture and the developmental sequence and time course are reflective of that described for PNs in vivo, synapse formation is initiated at an early stage of PN development in culture and proceeds concurrently with the morphological development, and the main period of synapse formation is associated with the main period of dendritic development, reflecting the preferential location of synaptic sites at the dendritic region of mature PN. Using electrophysiological techniques, we have examined the physiological development of PNs in culture and have correlated the stages in physiological, morphological, and synaptic development. Results from these studies show the following. Mature PNs in culture exhibit complex electrophysiological properties, including the ability to generate 2 types of spike events, simple and complex spikes, and endogenously generated activity. Expression of electrophysiological properties begins at an early stage in PN development, when the PNs consist of little more than a soma with a few fine perisomatic processes. The earliest physiological characteristics to be expressed by the PN include sensitivity to transmitters, the ability to respond to synaptic input, and the ability to generate simple spikes. Synaptic input produces spontaneous activity in young PNs, but the patterns of activity change during development as mechanisms underlying endogenously generated activity and complex spike generation are expressed, and synapse formation proceeds.(ABSTRACT TRUNCATED AT 400 WORDS)