Pilocarpine-induced acute seizure causes rapid area-specific astrogliosis and alters purinergic signaling in rat hippocampus.

The progressive nature of acquired epilepsy warrants a thorough examination of acute changes that occur immediately after an epileptogenic insult to better understand the cellular and molecular mechanisms that trigger epileptogenesis. Astrocytes are important regulators of neuronal functions and emerging evidence suggests an involvement of astrocytic purinergic signaling in the etiology of acquired epilepsy. However, how astrocytic purinergic signaling responds immediately after an acute seizure or an epileptogenic insult to impact epileptogenesis is not well studied. In the present study, we report area-specific rapid onset of astrocytic changes in morphology, as well as in expression and functional activity of the purinergic signaling in the hippocampus that occur immediately after pilocarpine-induced stage 5 seizure. After 3 hr of stage 5 acute seizure, hippocampal astrocytes show increased intrinsic calcium activity in stratum radiatum as well as reactive astrogliosis in the stratum lacunosum moleculare and hilus regions of the hippocampus. Hilar astrocytes also upregulated the expression of P2Y1 and P2Y2 metabotropic purinergic receptors. Subsequently, P2Y1 exhibited a functional upregulation by showing a significantly higher intracellular calcium rise in ex-vivo hippocampal slices on P2Y1 activation. Our results suggest that hippocampal astrocytes undergo rapid area-specific morphological and functional changes immediately after the commencement of the seizure activity and purinergic receptors upregulation is one of the earliest changes in response to seizure activity. These changes can be considered acute astrocytic responses to seizure activity which can potentially drive the epileptogenesis and can be explored further to identify astrocyte-specific targets for seizure therapy.

Picture perfect: Imaging mitochondrial membrane potential changes in retina slices with minimal stray fluorescence.

Mitochondrial membrane potential (Ψm) is a critical parameter that can be used to determine cellular well-being. As it is a direct measure of the cell's ATP generating capability, in recent years, this key component in cell biology has been the subject of thousands of biochemical and biophysical investigations. Membrane-permeant fluorescent dyes, like tetramethylrhodamine ethyl ester (TMRE), have been predominantly employed to monitor ΔΨm in cells. These dyes are typically lipophilic cationic compounds that equilibrate across membranes in a Nernstian fashion, thus accumulating into the mitochondrial membrane matrix space in inverse proportion to Ψm. However, the bath loading method practiced for labelling tissue slices with these cationic dyes poses limitations in the form of non-specificity and low signal to noise ratio, which compromises the precision of the results. Therefore, we introduce an alternative way for TMRE loading to image the ΔΨm in tissue slices by utilizing a low resistance glass pipette attached to a pressure injector. This method shows highly precise fluorescent dye labelling of the mitochondria and offers maximum output intensity, in turn enhancing signal to noise ratio.

Early Diabetes Induces Changes in Mitochondrial Physiology of Inner Retinal Neurons.

Diabetic retinopathy, a leading cause of vision loss, was considered as a solely vascular disorder but some recent studies suggest that retinal neurons may be affected much before the appearance of vascular lesions. However, the cellular processes involved in diabetes-induced degeneration of retinal neurons are poorly understood. Calcium (Ca2+) signaling plays a key role in normal functioning of neurons, and its dysregulation may lead to degeneration of neurons. Mitochondria are crucial components involved in the regulation of intracellular Ca2+ signaling. In this study, we have investigated the effects of diabetes on Ca2+ signaling in retinal neurons. The study was performed in rat retinal neurons cultured in high glucose condition (HGC) for 7-14 days and in acutely prepared retinal slices isolated from diabetic rats. When Ca2+ influx was induced by depolarization of neurons with 60 mM KCl in HGC neurons, the Ca2+ rise was sustained for a much longer duration as compared to controls, suggesting perturbation of Ca2+ buffering. In addition, HGC neurons also showed notably enhanced Ca2+ load in the mitochondria, which was accompanied by depolarization of mitochondrial membrane and enhanced reactive oxygen species formation. Similar results were obtained in acutely prepared retinal slices from control and diabetic rats. The depolarization of mitochondrial membrane was more pronounced in the neurons of the inner nuclear layer of diabetic rats. The physiological changes in mitochondria were observed as early as 9 weeks post diabetes induction. Thus, we report here that the intracellular Ca2+ signaling and mitochondrial function in retinal neurons are altered at an early stage of diabetes.

AMPA receptor activation causes preferential mitochondrial Ca²âº load and oxidative stress in motor neurons.

It is well established that motor neurons are highly vulnerable to glutamate induced excitotoxicity. The selective vulnerability of these neurons has been attributed to AMPA receptor mediated excessive rise in cytosolic calcium and consequent mitochondrial Ca(2+) loading. Earlier we have reported that in motor neurons a generic rise in [Ca(2+)]i does not always lead to mitochondrial Ca(2+) loading and membrane depolarization but it occurs upon AMPA receptor activation. The mechanism of such specific mitochondrial involvement upon AMPA receptor activation is not known. The present study examines the mitochondrial Ca(2+) regulation and oxidative stress in spinal cord neurons upon AMPA subtype of glutamate receptor activation. Stimulating the spinal neurons with AMPA exhibited a sharp rise in [Ca(2+)]m in both motor and other spinal neurons that was sustained up to the end of recording time of 30min. The rise in [Ca(2+)]m was substantially higher in motor neurons than in other spinal neurons which could be due to the differential mitochondrial homeostasis in two types of neurons. To examine this possibility, we measured AMPA induced [Ca(2+)]m loading in the presence of mitochondrial inhibitors. In both cell types the AMPA induced [Ca(2+)]m loading was blocked by mitochondrial calcium uniporter blocker ruthenium red. In motor neurons it was also inhibited substantially by CGP37157 and cyclosporine-A, the blockers of Na(+)/Ca(2+) exchanger and mitochondrial permeability transition pore (MPTP) respectively, whereas no effect of these agents was observed in other spinal neurons. Thus in motor neurons the Ca(2+) sequestration by mitochondria occurs through mitochondrial calcium uniporter as well as due to reversal of Na(+)/Ca(2+) exchanger, in contrast the latter pathway does not contribute in other spinal neurons. The ROS formation was inhibited by nitric oxide synthase (NOS) inhibitor L-NAME in both types of neurons, however the mitochondrial complex-I inhibitor rotenone suppressed the ROS formation only in motor neurons. It appears that activation of cytoplasmic nNOS leads to ROS formation in both types of spinal neurons but mitochondria is the major source of ROS in motor neurons. Spinal neurons exhibited a significant time dependent fall in glutathione (GSH) level. The GSH level in motor neurons did not recover even at 24h after AMPA exposure, whereas the other spinal neurons exhibited a tendency to maintain the GSH after a certain level suggesting that the oxidative stress is arrested in other spinal neurons but it continues to increase in motor neurons. Thus our results demonstrate that upon AMPA receptor stimulation the motor neurons employ some additional pathways for regulation of mitochondrial calcium and oxidative stress as compared to other spinal neurons. It is suggested that such differential signaling mechanisms in motor neurons could be crucial for their selective vulnerability to excitotoxicity.

Depalmitoylation preferentially downregulates AMPA induced Ca2+ signaling and neurotoxicity in motor neurons.

Excessive activation of AMPA receptor has been implicated in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). However, it is not clear why motor neurons are preferentially sensitive to AMPA receptor mediated excessive [Ca(2+)]i rise and excitotoxicity. In the present study we examined whether palmitoylation regulates Ca(2+) permeability of AMPA receptor and excitotoxicity in cultured spinal cord neurons. We adapted chronic 2-bromopalmitate (2-BrP) treatment to achieve depalmitoylation and examined its effect on the cytotoxicity in spinal cord neurons exposed to AMPA. The change in AMPA induced signaling and cytotoxicity in motor neurons and other spinal neurons under identical conditions of exposure to AMPA was studied. 2-BrP treatment inhibited AMPA induced rise in [Ca(2+)]i and cytotoxicity in both types of neurons but the degree of inhibition was significantly higher in motor neurons as compared to other spinal neurons. The AMPA induced [Na(+)]i rise was moderately affected in both type of neurons on depalmitoylation. Depalmitoylation reduced the expression levels of AMPA receptor subunits (GluR1 and GluR2) and also PSD-95 but stargazin levels remained unaffected. Our results demonstrate that 2-BrP attenuates AMPA receptor activated Ca(2+) signaling and cytotoxicity preferentially in motor neurons and suggest that AMPA receptor modulation by depalmitoylation could play a significant role in preventing motor neuron degeneration.

AMPA induced Ca2+ influx in motor neurons occurs through voltage gated Ca2+ channel and Ca2+ permeable AMPA receptor.

The rise in intracellular Ca(2+) mediated by AMPA subtype of glutamate receptors has been implicated in the pathogenesis of motor neuron disease, but the exact route of Ca(2+) entry into motor neurons is not clearly known. In the present study, we examined the role of voltage gated calcium channels (VGCCs) in AMPA induced Ca(2+) influx and subsequent intracellular signaling events responsible for motor neuron degeneration. AMPA stimulation caused sodium influx in spinal neurons that would depolarize the plasma membrane. The AMPA induced [Ca(2+)](i) rise in motor neurons as well as other spinal neurons was drastically reduced when extracellular sodium was replaced with NMDG, suggesting the involvement of voltage gated calcium channels. AMPA mediated rise in [Ca(2+)](i) was significantly inhibited by L-type VGCC blocker nifedipine, whereas ω-agatoxin-IVA and ω-conotoxin-GVIA, specific blockers of P/Q type and N-type VGCC were not effective. 1-Napthyl-acetyl spermine (NAS), an antagonist of Ca(2+) permeable AMPA receptors partially inhibited the AMPA induced [Ca(2+)](i) rise but selectively in motor neurons. Measurement of AMPA induced currents in whole cell voltage clamp mode suggests that a moderate amount of Ca(2+) influx occurs through Ca(2+) permeable AMPA receptors in a subpopulation of motor neurons. The AMPA induced mitochondrial calcium loading [Ca(2+)](m), mitochondrial depolarization and neurotoxicity were also significantly reduced in presence of nifedipine. Activation of VGCCs by depolarizing concentration of KCl (30mM) in extracellular medium increased the [Ca(2+)](i) but no change was observed in mitochondrial Ca(2+) and membrane potential. Our results demonstrate that a subpopulation of motor neurons express Ca(2+) permeable AMPA receptors, however the larger part of Ca(2+) influx occurs through L-type VGCCs subsequent to AMPA receptor activation and consequent mitochondrial dysfunction is the trigger for motor neuron degeneration. Nifedipine is an effective protective agent against AMPA induced mitochondrial stress and degeneration of motor neurons.

Neuronal plasticity of human Wharton's jelly mesenchymal stromal cells to the dopaminergic cell type compared with human bone marrow mesenchymal stromal cells.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) derived from Wharton's jelly (WJ) of the umbilical cord are increasingly gaining prominence as substitutes for bone marrow (BM) MSC. While MSC isolated from different tissue sources may share common mesenchymal properties, the difference in their plasticity to individual lineages is ill-defined. Thus the focus of this study was to estimate the neuronal plasticity of WJ MSC to the dopaminergic (DA) cell type in comparison with BM MSC. METHODS: For neuronal differentiation, MSC were exposed to developmentally relevant cues for midbrain DA neurons: sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8), along with basic fibroblast growth factor (bFGF). RESULTS: Naive MSC from both sources constitutively expressed neuronal markers. Flow cytometry data revealed that the control WJ MSC shared a signature similar to BM MSC for early neuronal markers (nestin, musashi12 and A2B5) and DA-specific markers [tyrosine hydroxylase (TH) and Nuclear Receptor related protein 1 (Nurr1) but differed for mature neuronal proteins [ß-tubulin III and microtubule-associated protein 2 (Map2ab)]. Similar populations of cells in both sources of MSC were positive for the SHH receptors [patched (PTCH) and smoothened (SMO)]. In induced BM and WJ MSC, real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis showed similar levels of DA-related transcription factors Nurr1 and Engrailed (En) 1. Immunocytochemical and flow cytometry analysis showed an increase in mature neuronal marker Map2ab. Kv4.2, a K(+) channel marker, was observed only in the induced MSC. Induced MSC also expressed several DA-specific markers, TH, dopamine and cyclic AMP regulated phosphoprotein (DARPP) 32, paired-like homeodomain transcription factor (PitX) 3 and vesicular monoamine transporter (VMAT) 2, in comparable levels between the two sources. The efficiency (c. 65%) of transdifferentiation of WJ MSC to TH-positive cells was similar to that of induced BM MSC. Constitutive and inducible release of dopamine was found to be similar between induced BM and WJ MSC, as measured by dopamine enzyme-linked immunosorbent assay (ELISA). Interestingly, an adenosine triphosphate (ATP)-stimulated change in intracellular Ca(2+) was observed in both control and induced MSC, but only the induced MSC was capable of releasing dopamine. CONCLUSIONS: Our data demonstrate that MSC from the two different sources respond similarly to inductive cues to differentiate terminally to a DA cell type, and the neuronal plasticity of human WJ MSC is comparable with that of BM MSC.

Chlorin p6 preferentially localizes in endoplasmic reticulum and Golgi apparatus and inhibits Ca(2+) release from intracellular store.

Subcellular localization of chlorin p6 in human cerebral glioma (U-87MG) cells was studied using laser scanning confocal microscopy. Localization in sub cellular organelles was ascertained by double labeling with specific fluorescent markers of subcellular organelles. The results reveal that chlorin p6 binds to multiple cellular sites but preferential binding sites are endoplasmic reticulum and Golgi apparatus and it does not bind to mitochondria. Significantly the drug localization pattern of proliferating and differentiated cells was notably distinct. In proliferating cells the internalization of drug was faster than in differentiated cells. Localization of chlorin p6 into the cells inhibited Ca(2+) release from endoplasmic reticulum and deregulated cellular Ca(2+) signalling. These results suggest that the fluorescence imaging pattern of chlorin p6 could be useful in identifying the proliferating and differentiated population of cells in tumor tissue.

Nitric oxide-mediated modulation of synaptic activity by astrocytic P2Y receptors.

We investigated the mechanism of synaptic suppression by P2Y receptors in mixed hippocampal cultures wherein networked neurons exhibit synchronized Ca(2+) oscillations (SCO) due to spontaneous glutamatergic synaptic transmission. Pharmacological studies suggested that SCO suppression was mediated by P2Y2/P2Y4 receptors. Immunostaining studies and characterization of ATP/UTP-stimulated Ca(2+) responses in solitary neurons and astrocytes revealed that the SCO attenuation was effectuated by astrocytes. We demonstrate that nitric oxide released from activated astrocytes causes synaptic suppression by inhibiting neurotransmitter release. Physiological concentrations of ATP and UTP evoked NO production in astrocytes. SCO suppression was considerably diminished by removal of extracellular NO by membrane-impermeable scavenger c-PTIO or by pretreatment of cells with nitric oxide synthase inhibitor L-NAME. The nitric oxide donor DETA/NO effectively suppressed the SCO. ATP/UTP inhibited KCl-induced exocytosis at presynaptic terminals in an NO-dependent manner. In the absence of exogenously added ATP/UTP, both the NO scavenger and NOS inhibitor enhanced the frequency of SCO, implying that astrocytes release NO during spontaneous synaptic activity and exert a suppressive effect. We report for the first time that under physiological conditions astrocytes use NO as a messenger molecule to modulate the synaptic strength in the networked neurons.

Knockdown of cytosolic glutaredoxin 1 leads to loss of mitochondrial membrane potential: implication in neurodegenerative diseases.

Mitochondrial dysfunction including that caused by oxidative stress has been implicated in the pathogenesis of neurodegenerative diseases. Glutaredoxin 1 (Grx1), a cytosolic thiol disulfide oxido-reductase, reduces glutathionylated proteins to protein thiols and helps maintain redox status of proteins during oxidative stress. Grx1 downregulation aggravates mitochondrial dysfunction in animal models of neurodegenerative diseases, such as Parkinson's and motor neuron disease. We examined the mechanism underlying the regulation of mitochondrial function by Grx1. Downregulation of Grx1 by shRNA results in loss of mitochondrial membrane potential (MMP), which is prevented by the thiol antioxidant, alpha-lipoic acid, or by cyclosporine A, an inhibitor of mitochondrial permeability transition. The thiol groups of voltage dependent anion channel (VDAC), an outer membrane protein in mitochondria but not adenosine nucleotide translocase (ANT), an inner membrane protein, are oxidized when Grx1 is downregulated. We then examined the effect of beta-N-oxalyl amino-L-alanine (L-BOAA), an excitatory amino acid implicated in neurolathyrism (a type of motor neuron disease), that causes mitochondrial dysfunction. Exposure of cells to L-BOAA resulted in loss of MMP, which was prevented by overexpression of Grx1. Grx1 expression is regulated by estrogen in the CNS and treatment of SH-SY5Y cells with estrogen upregulated Grx1 and protected from L-BOAA mediated MMP loss. Our studies demonstrate that Grx1, a cytosolic oxido-reductase, helps maintain mitochondrial integrity and prevents MMP loss caused by oxidative insult. Further, downregulation of Grx1 leads to mitochondrial dysfunction through oxidative modification of the outer membrane protein, VDAC, providing support for the critical role of Grx1 in maintenance of MMP.

NMDA and non-NMDA receptor-mediated differential Ca2+ load and greater vulnerability of motor neurons in spinal cord cultures.

Glutamate receptor activated neuronal cell death has been implicated in the pathogenesis of motor neuron disease but the molecular mechanism responsible for neuronal dysfunction needs to be elucidated. In the present study, we examined the contribution of NMDA and non-NMDA sub-types of glutamate receptors in selective vulnerability of motor neurons. Glutamate receptor activated Ca2+ signaling, mitochondrial functions and neurotoxicity in motor neurons and other spinal neurons were studied in mixed spinal cord primary cultures. Exposure of cells to glutamate receptor agonists glutamate, NMDA and AMPA elevated the intracellular Ca2+, mitochondrial Ca2+ and caused mitochondrial depolarization and cytotoxicity in both motor neurons and other spinal neurons but a striking difference was observed in the magnitude and temporal patterns of the [Ca2+]i responses between the two neuronal cell types. The motor neurons elicited higher Ca2+ load than the other spinal neurons and the [Ca2+]i levels were elevated for a longer duration in motor neurons. AMPA receptor stimulation was more effective than NMDA. Both the NMDA and non-NMDA receptor antagonists APV and NBQX inhibited the Ca2+ entry and decreased the cell death significantly; however, NBQX was more potent than APV. Our results demonstrate that both NMDA and non-NMDA sub-types of glutamate receptors contribute to glutamate-mediated motor neuron damage but AMPA receptors play the major role. AMPA receptor-mediated excessive Ca2+ load and differential handling/regulation of Ca2+ buffering by mitochondria in motor neurons could be central in their selective vulnerability to excitotoxicity.

Lipid raft heterogeneity: an enigma.

Glycosphingolipids (GSLs) and glycoproteins are ubiquitous components of mammalian cell membranes. GSLs are especially enriched in the nervous system and significantly contribute to membrane organization and a variety of cellular functions. Current body of evidence suggests that GSLs along with cholesterol are enriched in discrete membrane domains that associate specific proteins. Current notion of membrane organization is that, the GSL-cholesterol-enriched membrane domains known as 'lipid rafts' float in the phospholipid-enriched bulk of the membrane and regulate the cell signaling by facilitating the lipid-protein/protein-protein interactions. The sizeable literature accumulated during the last decade has provided some insight into the organization and function of rafts; however, they still remain perplexing. In recent years, an appealing concept of lipid raft heterogeneity has emerged. GSL- and glycosylphosphatidylinositol-anchored proteins are considered as the crucial pivots of heterogeneous rafts. This review deals with the enigma of organizational and functional heterogeneity of lipid rafts and discusses the dynamic coalescence of heterogeneous rafts during signaling that can explain the specificity of raft-regulated cellular signaling events.

Melanocyte-keratinocyte interaction induces calcium signalling and melanin transfer to keratinocytes.

Physical contact between melanocytes and keratinocytes is a prerequisite for melanosome transfer to occur, but cellular signals induced during or after contact are not fully understood. Herein, it is shown that interactions between melanocyte and keratinocyte plasma membranes induced a transient intracellular calcium signal in keratinocytes that was required for pigment transfer. This intracellular calcium signal occurred due to release of calcium from intracellular stores. Pigment transfer observed in melanocyte-keratinocyte co-cultures was inhibited when intracellular calcium in keratinocytes was chelated. We propose that a 'ligand-receptor' type interaction exists between melanocytes and keratinocytes that triggers intracellular calcium signalling in keratinocytes and mediates melanin transfer.

NMDA and non-NMDA receptors stimulation causes differential oxidative stress in rat cortical slices.

Glutamate receptor activated neuronal cell death is attributed to a massive influx of Ca(2+) and subsequent formation of reactive oxygen species (ROS) but the relative contribution of NMDA and non-NMDA sub-types of glutamate receptors in excitotoxicity is not known. In the present study, we have examined the role of NMDA and non-NMDA receptors in glutamate-induced neuronal injury in cortical slices from young (20+/-2 day) and adult (80+/-5 day) rats. Treatment of slices with glutamate receptor agonists NMDA, AMPA and KA elicited the formation of reactive oxygen species (ROS) and neuronal cell death. In young slices, NMDA receptor stimulation caused a higher ROS formation and neurotoxicity, but KA was more effective in producing ROS and cell death in adult slices. AMPA exhibited an intermediate effect on ROS formation and toxicity in both the age groups. A significant protection in glutamate mediated ROS formation and neurotoxicity was observed in presence of NMDA or/and non-NMDA receptors antagonists APV and NBQX, respectively. This further confirms the involvement of both NMDA and non-NMDA receptors in glutamate mediated neurotoxicity. In adult slices, we did not find positive correlation between ligand induced neurotoxicity and mitochondrial depolarization. Though, NMDA and KA stimulation produced differential effect on ROS formation and neurotoxicity in young and adult slices, the mitochondrial depolarization was higher and comparable on NMDA stimulation in both the age groups as compared to KA, suggesting that the mitochondrial depolarization may not be a good indicator for neurotoxicity. Our results demonstrate that both NMDA and non-NMDA sub-types of glutamate receptors are involved in glutamate mediated neurotoxicity but their relative contribution is highly dependent on the age of the animal.

Co-activation of P2Y2 receptor and TRPV channel by ATP: implications for ATP induced pain.

1. Extracellular ATP is recognized as a peripheral modulator of pain. Activation of ionotropic P2X receptors in sensory neurons has been implicated in induction of pain, whereas metabotropic P2Y receptors in potentiation of pain induced by chemical or physical stimuli via capsaicin sensitive TRPV1 channel. Here we report that P2Y2 receptor activation by ATP can activate the TRPV1 channel in absence of any other stimuli. 2. ATP-induced Ca2+ signaling was studied in Neuro2a cells. ATP evoked release of intracellular Ca2+ from ER and Ca2+ influx through a fast inactivating channel. The Ca2+ response was induced by P2Y receptor agonists in the order of potency ATP>or=UTP>or=ATPgammaS>ADP and was inhibited by suramin and PPADS. The P2X receptor agonist alpha beta methyl ATP was ineffective. 3. The Ca2+ influx was blocked by ruthenium red, an inhibitor of TRPV1 channel. Capsaicin, the most potent activator of the TRPV1 channel, evoked a fast inactivating Ca2+ transient suggesting the presence of endogenous TRPV1 channels in Neuro2a cells. NMS and PDBu, repressors of IP3 formation, drastically inhibited both the components of Ca2+ response. 4. Our data show co-activation of the P2Y2 receptor and capsaicin sensitive TRPV1 channel by ATP. Such functional interaction between endogenous P2Y2 receptor and TRPV1 channels could explain the ATP-induced pain.

Cerebrospinal fluid from amyotrophic lateral sclerosis patients preferentially elevates intracellular calcium and toxicity in motor neurons via AMPA/kainate receptor.

Several lines of evidence in the literature purport the contribution of glutamate mediated excitotoxicity in the etiology of amyotrophic lateral sclerosis (ALS) but the cellular mechanisms responsible for selective loss of motor neurons are still obscure. Elevation of intracellular Ca(2+) is considered as the early event in glutamate mediated cell injury. We have studied the changes in [Ca(2+)](i) and cytotoxicity in motor neurons and other spinal neurons in culture upon exposure to cerebrospinal fluid (CSF) from ALS patients. CSFs from 20 ALS patients and 20 disease control patients were examined. Eighteen out of twenty (90%) ALS-CSF samples induced a transient but pronounced elevation of [Ca(2+)](i) in neurons, whereas only 1/20 (5%) sample from disease control patients induced a marginal elevation of [Ca(2+)](i). Strikingly the [Ca(2+)](i) rise was 2-3-fold higher and longer lasting in motor neurons in comparison to the other spinal neurons. Exposure of cells to ALS-CSF drastically decreased the survival rate of motor neurons to 32.26+/-2.06% whereas a moderate decrease was observed in case of other spinal neurons (67.90+/-2.04%). In cultures treated with disease control CSF, a small decrease was observed in the survival rate with 80.14+/-2.00% and 90.07+/-1.37% survival of motor neuron and other spinal neurons respectively. The AMPA/kainate receptor antagonist NBQX rendered significant protection against the ALS-CSF induced Ca(2+) influx and neurotoxicity while the NMDA receptor antagonist APV showed a mild effect. Our data demonstrate that the exposure of spinal cord neurons to ALS-CSF differentially elevates [Ca(2+)](i) and neurotoxicity in motor neurons by activation of glutamate receptors, the AMPA/kainate receptor playing the major role.

Xanthine oxidase, nitric oxide synthase and phospholipase A(2) produce reactive oxygen species via mitochondria.

The formation of reactive oxygen species (ROS) has been suggested to be associated with excitotoxicity but the involvement of cytoplasmic enzymes in ROS formation is not clearly known. In the present study, we examined the role of xanthine oxidase (XO), nitric oxide synthase (NOS) and phospholipase A(2) (PLA(2)) in glutamate-induced oxidative stress in rat cortical slices. Glutamate-induced ROS formation and mitochondrial depolarization were measured in rat cortical slices in presence of allopurinol, L-NAME and 4-bromophenacylbromide, the specific inhibitors of XO, NOS and PLA(2), respectively. Upon stimulation of slices with glutamate, a significant increase in ROS formation and mitochondrial depolarization was observed. However, pretreatment of slices with allopurinol, L-NAME and 4-bromophenacylbromide inhibited the glutamate-induced ROS formation and mitochondrial depolarization. The glutamate-induced ROS formation was dependent on the concentration of these inhibitors and also on the duration of the treatment. Allopurinol was found to be less effective as compared to L-NAME and 4-bromophenacylbromide. The combined treatment of slices with these enzyme inhibitors showed further inhibition in ROS formation and mitochondrial depolarization. The inhibition in ROS formation as well as mitochondrial depolarization by allopurinol, L-NAME and 4-bromophenacylbromide clearly suggests that the activation of XO, NOS and PLA(2) by calcium during glutamate receptor stimulation may release some chemicals which depolarize mitochondria resulting in ROS formation.

Glutamate-induced differential mitochondrial response in young and adult rats.

Excitatory amino acid glutamate is involved in neurotransmission in the nervous system but it becomes a potent neurotoxin under variety of conditions. However, the molecular mechanism of excitotoxicity is not known completely. We have studied the influence of glutamate on intracellular calcium and mitochondrial functions in cortical slices from young and adult rats. The slices from both the age groups exhibited comparable intracellular calcium changes upon glutamate stimulation. Glutamate treatment caused a decrease in adenosine 5'-diphosphate/adenosine 5'-triphosphate (ADP/ATP) and an increase in nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide reduced form (NAD/NADH) ratio in both the age groups but the magnitude and the nature of temporal change was different. Glutamate-induced decrease in ATP/ADP and increase in NAD/NADH ratio was significantly higher in slices from the adult as compared to the young rats. The slices from young rats elicited slightly higher mitochondrial depolarization than adult rats. However, the formation of reactive oxygen species (ROS) and lactate dehydrogenase (LDH) release were significantly higher in adult rats as compared to young rats. Glutamate-induced mitochondrial depolarization, ROS formation and LDH release were highly dependent on the presence of Ca(2+) in the extracellular medium. The treatment of slices with mitochondrial inhibitors rotenone and oligomycin inhibited ROS formation and LDH release substantially. Our results suggest that the glutamate-induced increase in intracellular calcium is not the only factor responsible for neuronal cell death but the mitochondrial functions could be crucial in excitotoxicity.

Liquid-ordered microdomains in lipid rafts and plasma membrane of U-87 MG cells: a time-resolved fluorescence study.

Lipid rafts, the functional microdomains in the cell membrane, are believed to exist as liquid-ordered (Lo) phase domains along with the liquid-disordered (Ld) phase of the bulk of the cell membranes. We have examined the lipid order in model and natural membranes by time-resolved fluorescence of trimethylammonium-1,6-diphenylhexatriene incorporated into the membranes. The lipid phases were discerned by the limiting anisotropy, rotational diffusion rate and distribution of the fluorescence lifetime. In dipalmitoylphosphatidylcholine (DPPC)-cholesterol mixtures the gel phase exhibited higher anisotropy and a two-fold slower rotational diffusion rate of the probe as compared to the Ld phase. On the other hand, the Lo phase exhibited higher limiting anisotropy but a rotational diffusion rate comparable to the Ld phase. The Ld and Lo phases elicited unimodal distribution of lifetimes with distinct mean values and their co-existence in phospholipid-cholesterol mixtures was reflected as a biphasic change in the width of the lifetime distribution. Global analysis of the lifetimes yielded a best fit with two lifetimes which were identical to those observed in single Lo or Ld phases, but their fractional contribution varied with cholesterol concentration. Attributing the shorter and longer lifetime components to the Ld and Lo phases, respectively, the extent of the Lo/Ld phase domains in the membranes was estimated by their fractional contribution to the fluorescence decay. In ternary mixtures of egg PC-gangliosides-cholesterol, the gangliosides induced heterogeneity in the membrane but the Ld phase prevailed. The Lo phase properties were observed only in the presence of cholesterol. Results obtained in the plasma membrane and detergent-resistant membrane fractions (DRMs) isolated from U-87 MG cells revealed that DRMs mainly possess the Lo phase; however, a substantially large proportion of plasma membrane also exists in the Lo phase. Our data show that, besides cholesterol, the membrane proteins play a significant role in the organization of lipid rafts and, furthermore, a considerable amount of heterogeneity is present among the lipid rafts.