Primary Cultures Of Rat Striatum Research Articles

Nanosize Titanium Dioxide Stimulates Reactive Oxygen Species in Brain Microglia and Damages Neurons in Vitro

BackgroundTitanium dioxide is a widely used nanomaterial whose photo-reactivity suggests that it could damage biological targets (e.g., brain) through oxidative stress (OS).ObjectivesBrain cultures of immortalized mouse microglia (BV2), rat dopaminergic (DA) neurons (N27), and primary cultures of embryonic rat striatum, were exposed to Degussa P25, a commercially available TiO2 nanomaterial. Physical properties of P25 were measured under conditions that paralleled biological measures.FindingsP25 rapidly aggregated in physiological buffer (800–1,900 nm; 25°C) and exposure media (~ 330 nm; 37°C), and maintained a negative zeta potential in both buffer (–12.2 ± 1.6 mV) and media (–9.1 ± 1.2 mV). BV2 microglia exposed to P25 (2.5–120 ppm) responded with an immediate and prolonged release of reactive oxygen species (ROS). Hoechst nuclear stain was reduced after 24-hr (≥100 ppm) and 48-hr (≥2.5 ppm) exposure. Microarray analysis on P25-exposed BV2 microglia indicated up-regulation of inflammatory, apoptotic, and cell cycling pathways and down-regulation of energy metabolism. P25 (2.5–120 ppm) stimulated increases of intracellular ATP and caspase 3/7 activity in isolated N27 neurons (24–48 hr) but did not produce cytotoxicity after 72-hr exposure. Primary cultures of rat striatum exposed to P25 (5 ppm) showed a reduction of immunohistochemically stained neurons and microscopic evidence of neuronal apoptosis after 6-hr exposure. These findings indicate that P25 stimulates ROS in BV2 microglia and is nontoxic to isolated N27 neurons. However, P25 rapidly damages neurons at low concentrations in complex brain cultures, plausibly though microglial generated ROS.

Amyloid β‐peptide(1–42) and hydrogen peroxide‐induced toxicity are mediated by TRPM2 in rat primary striatal cultures

Amyloid beta-peptide (Abeta) is the main component of senile plaques which characterize Alzheimer's disease and may induce neuronal death through mechanisms which include oxidative stress. To date, the signalling pathways linking oxidant stress, a component of several neurodegenerative diseases, to cell death in the CNS are poorly understood. Melastatin-like transient receptor potential 2 (TRPM2) is a Ca(2+)-permeant non-selective cation channel, which responds to increases in oxidative stress levels in the cell and is activated by oxidants such as hydrogen peroxide. We demonstrate here that Abeta and hydrogen peroxide both induce death in cultured rat striatal cells which express TRPM2 endogenously. Transfection with a splice variant that acts as a dominant negative blocker of TRPM2 function (TRPM2-S) inhibited both hydrogen peroxide- and Abeta-induced increases in intracellular-free Ca(2+) and cell death. Functional inhibition of TRPM2 activation by the poly(ADP-ribose)polymerase inhibitor SB-750139, a modulator of intracellular pathways activating TRPM2, attenuated hydrogen peroxide- and Abeta-induced cell death. Furthermore, a small interfering RNA which targets TRPM2, reduced TRPM2 mRNA levels and the toxicity induced by hydrogen peroxide and Abeta. These data demonstrate that activation of TRPM2, functionally expressed in primary cultures of rat striatum, contributes to Abeta- and oxidative stress-induced striatal cell death.

Dopamine-dependent increases in phosphorylation of cAMP response element binding protein (CREB) during precipitated morphine withdrawal in primary cultures of rat striatum.

Chronic morphine leads to compensatory up-regulation of cAMP signaling pathways in numerous brain regions. One potential consequence of up-regulated cAMP signaling is increased phosphorylation of cAMP response element binding protein (CREB), a transcription factor that may regulate neuroadaptations related to morphine dependence. Altered gene expression within the nucleus accumbens (NAc), a ventral component of the striatum that receives substantial dopaminergic input, may play a role in some of the motivational aspects of opiate withdrawal. To determine if morphine withdrawal leads to increased CREB phosphorylation in striatal tissues, we examined the effects of naloxone-precipitated morphine withdrawal on CREB phosphorylation in primary cultures of rat striatal neurons. Precipitated morphine withdrawal was associated with enhanced dopamine-, SKF 82958 (D1 receptor agonist)-, and forskolin-induced CREB phosphorylation. During precipitated withdrawal, D1 receptor-mediated CREB phosphorylation was dependent on cAMP-dependent protein kinase (PKA). Precipitated withdrawal also led to up-regulation of c-fos mRNA in response to SKF 82958. CREB protein levels were not altered by acute or chronic morphine. These results suggest that D1 receptor-mediated signal transduction is enhanced during morphine withdrawal. Furthermore, they are consistent with in vivo evidence suggesting that increased CREB activation in portions of the striatum (e.g. the NAc) is related to dysphoric states associated with drug withdrawal.

Molecular Components of Striatal Plasticity: The Various Routes of Cyclic AMP Pathways

Neuroplasticity serves an important role for normal striatal function and in disease states. One route to neuroplasticity involves activation of the transcription factor cyclic 3′,5′-adenosine monophosphate (cyclic AMP) response element binding protein (CREB) by phosphorylation of the amino acid ¹³³Ser. Dopamine and glutamate, the two predominant neurotransmitters in the striatum, induce CREB phosphorylation in primary cultures of rat striatum through cyclic AMP and Ca²⁺ pathways. Here we present the role of N-methyl-D-aspartate receptors and Ca²⁺ in cyclic AMP-mediated CREB phosphorylation.

Suppression of synaptic exocytosis through 5-HT 1A receptor in cultured rat hippocampal neuron

The neurotoxicity of l-DOPA and dopamine (DA) on striatal neurons was examined by using primary cultures of rat striatum. Exposure to l-DOPA and DA at concentrations of 30–300 μM induced dose-dependent cell death in both younger cultures (3 days in culture, 3 DIC) and elder cultures (10 days in culture, 10 DIC). The cytotoxicity of l-DOPA and DA was also dependent on the exposure time (6–24 h). Ascorbic acid (200 μM) inhibited both l-DOPA- and DA-induced cytotoxicity in 3 DIC cultures, whereas it provided significant protection against DA- but not l-DOPA-induced cytotoxicity in 10 DIC cultures. The l-DOPA cytotoxicity in 10 DIC cultures was prevented by a non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and by an NMDA receptor antagonist, MK-801. Neither antagonist prevented DA cytotoxicity. d-DOPA did not affect the viability of 10 DIC cultures, though it elicited marked toxicity in 3 DIC cultures. These results suggest that there are two components in the mechanisms that mediate the l-DOPA neurotoxicity on striatal neurons: one is autoxidation-relevant and the other is autoxidation-irrelevant. With respect to the latter, glutamate receptor stimulation may be involved. In contrast, autoxidation plays an important role in DA neurotoxicity.

Expression of NMDA Receptor-1 (NR1) and Huntingtin in Striatal Neurons Which Colocalize Somatostatin, Neuropeptide Y, and NADPH Diaphorase: A Double-Label Histochemical and Immunohistochemical Study

The subset of striatal neurons which colocalize SS/NPY/NADPH-d are selectively resistant to neurodegeneration in Huntington's Disease (HD) and to excitotoxic cell death induced experimentally with NMDA receptor (NMDAR) agonists. Here we have analyzed the expression of immunoreactive NMDAR-1 (NR1) subunit (as an index of NMDAR protein) and of huntingtin (the normal product of the HD gene) in primary cultures of rat striatum to see if differential expression of the two antigens in the subset of SS/NPY/NADPH-d and other striatal neurons can explain their selective resistance or vulnerability. Double-label histochemical and immunocytochemical studies were carried out using conventional and confocal laser scanning microscopy to characterize the cellular and subcellular expression of NR1 and SS, or NPY or bNOS, together with NADPH-d histochemistry. The percentages of cultured striatal neurons that were positive for NADPH-d, SS, NPY, bNOS, and NRI were, respectively, 3.8, 8.4, 10.2, 5.1, and 80%. The majority of striatal NADPH-d neurons coexpressed SS and NPY; 17% of SS-producing neurons were strongly positive for NR1; the remaining cells (≈80%) exhibited only weak NR1 expression. Comparable data were obtained for NPY-positive neurons, 15% of which colocalized NR1 strongly and 70–80% weakly. By double-label immunofluorescence, huntingtin was nonselectively expressed in virtually all striatal neurons including SS/NPY/NADPH-d neurons. These results show that the majority of striatal SS/NPY/NADPH-d neurons express NR1. The relative abundance of NR1 in SS/NPY/NADPH-d neurons, however, varies between a small subset of neurons that are receptor rich and the remainder that express low levels only and may determine susceptibility to NMDAR-mediated neurotoxicity. Huntingtin is nonselectively expressed in virtually all striatal neurons and does not appear to be a determinant of the selective resistance of normal striatal SS/NPY/NADPH-d neurons to NMDA toxicity.

Differential neurotoxicity induced by l-DOPA and dopamine in cultured striatal neurons

The neurotoxicity of l-DOPA and dopamine (DA) on striatal neurons was examined by using primary cultures of rat striatum. Exposure to l-DOPA and DA at concentrations of 30–300 μM induced dose-dependent cell death in both younger cultures (3 days in culture, 3 DIC) and elder cultures (10 days in culture, 10 DIC). The cytotoxicity of l-DOPA and DA was also dependent on the exposure time (6–24 h). Ascorbic acid (200 μM) inhibited both l-DOPA- and DA-induced cytotoxicity in 3 DIC cultures, whereas it provided significant protection against DA- but not l-DOPA-induced cytotoxicity in 10 DIC cultures. The l-DOPA cytotoxicity in 10 DIC cultures was prevented by a non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and by an NMDA receptor antagonist, MK-801. Neither antagonist prevented DA cytotoxicity. d-DOPA did not affect the viability of 10 DIC cultures, though it elicited marked toxicity in 3 DIC cultures. These results suggest that there are two components in the mechanisms that mediate the l-DOPA neurotoxicity on striatal neurons: one is autoxidation-relevant and the other is autoxidation-irrelevant. With respect to the latter, glutamate receptor stimulation may be involved. In contrast, autoxidation plays an important role in DA neurotoxicity.

Effects of ATP analogues and basic fibroblast growth factor on astroglial cell differentiation in primary cultures of rat striatum

We have used primary cultures of rat striatum to study the effects of ATP analogues on the elongation of astrocytic processes, a parameter of astroglial cell differentiation. Parallel studies were performed with basic fibroblast growth factor, a known regulator of astroglial cell function. After three days in culture, both the growth factor and αβ-methylene-ATP induced dramatic increases in the mean length of astrocytic processes/cell. For both agents, effects were dose-dependent. The effect of αβ-methylene-ATP was antagonized by the trypanoside suramin and mimicked by 2-methyl-thio-ATP, suggesting the involvement of a suramin-sensitive P2-purinoceptor. Neither an additive nor a synergistic effect between αβ-methylene-ATP and basic fibroblast growth factor on the elongation of processes was detected in cultures exposed to both agents. Indeed, an inhibition with respect to the effects induced by either agent alone was recorded, suggesting that the growth factor and the purine analogue can modulate astrocytic differentiation by activation of common intracellular pathways.It is concluded that, like basic fibroblast growth factor, ATP can promote the maturation of astrocytes towards a more differentiated phenotype characterized by the presence of longer astrocytic processes. These findings might have interesting implications for astroglial cell differentiation during brain development and for ischemia- and trauma-associated hypergliosis.

Basic fibroblast growth factor induces c- fos expression in primary cultures of rat striatum

Basic fibroblast growth factor (bFGF) is present in the rat striatum in vivo, where evidence suggests it may have a long-term trophic role in supporting the survival of striatal neurones. To examine the possibility that these effects of bFGF might be mediated by induction of neuronal gene expression, we have investigated the ability of bFGF to stimulate expression of the immediate-early gene c- fos in primary cultures of embryonic rat striatum. The basal levels of c- fos mRNA were low in both neurones and glia in culture. Application of 500 pM bFGF resulted, within 45 min, in a 11-fold increase in the c- fos hybridisation signal in the non-neuronal cells. No significant induction of c- fos mRNA was detected in the striatal neurones at this time. The induction in non-neuronal cells was blocked by the tyrosine kinase inhibitor genistein (100 μM), but not by its inactive structural analogue genistin (100 μM). These results represent a novel mechanism whereby bFGF can exert prolonged effects on striatal function, and indicate that the increases in striatal c- fos gene expression induced by bFGF occur primarily in non-neuronal cells.

Modulation of astroglial cell proliferation by analogues of adenosine and ATP in primary cultures of rat striatum

We have studied the possible purinoceptor-mediated modulation of astroglial cell proliferation in neuron-glia primary cultures obtained from rat corpus striatum. Cultures were grown for three days in the presence of either 2-chloro-adenosine or αβ-methylene-ATP (which behave as agonists of adenosine/ P1 and ATP/P2 purinoceptors, respectively), and then immunostained with an antibody to glial fibrillary acidic protein. 2-Chloro-adenosine decreased and αβ-methylene-ATP increased the number of astroglial cells in culture. For both derivatives, the effect was dose-dependent. The effect of αβ-methylene-ATP was antagonized by the trypanoside suramin, suggesting the involvement of a suramin-sensitive P2 purinoceptor, whereas the effect of 2-chloro-adenosine was not reversed by the P1 purinoceptor antagonist p-sulphonyl-phenyl-theophylline, implying the activation of a xanthine-insensitive adenosine purinoceptor subtype. In order to evaluate the extent of astrocyte proliferation in the presence of these two analogues, some cultures were incubated with bromodeoxyuridine for 24 h before fixing, and then double-immunostained for glial fibrillary acidic protein and bromodeoxyuridine. The percentage of bromodeoxyuridine positive astrocytes was significantly increased after exposure to both agents. It is therefore concluded that purines can modulate astroglial cells in opposite ways, inducing decreases or increases of cell number by activation of P1 and P2 purinoceptors, respectively. For the P2 purinoceptor-mediated effect, there was a quantitative correlation between the percentage of bromodeoxyuridine positive astrocytes and the cell number. For the P1 purinoceptor-mediated effect, no apparent correlation between these two parameters was found. This suggests the activation of independent effects, which involve other mechanisms besides the stimulation of DNA synthesis, and which eventually result in a reduction of cell number. The possible relevance of these findings to in vivo regulation of astrocyte cell function as well as in trauma- and ischaemia-associated hypergliosis is discussed.

μ-Opioid receptor-regulated adenylate cyclase activity in primary cultures of rat striatal neurons upon chronic morphine exposure

Rat striatal neurons cultured in serum-free, hormone-supplemented medium, were exposed to 10 μM morphine for several hours or days before intracellular cyclic AMP production was measured. Dopamine D 1 receptor- and β-adrenoceptor-stimulated cyclic AMP production were prefoundly increased upon morphine exposure (up to 150% of control). In contrast, cyclic AMP production induced by direct activation of the catalytic unit of adenylate cyclase with forskolin remained unaffected. Interestingly, the relative inhibitory effect of the μ-opioid receptor agonist [D-Ala 2,MePhe 4,Gly-ol 5]enkephalin (DAGO) on dopamine D 1 receptor-stimulated cyclic AMP production was unchanged after exposure to morphine. On the other hand, unlike μ-opioid receptors chronically exposed to morphine, β-adrenoceptors mediating activation of adenylate cyclase were rapidly desensitized upon prolonged exposure of the neurons to isoprenaline. It is suggested that tolerance to morphine may be caused by the fact that morphine is acting against up-regulated signal transduction mechanisms rather than by μ receptor desensitization. Moreover, this enhanced effector system function may be involved in opioid dependence. The adaptive changes following morphine treatment appear to be independent of possible alterations at the level of dopaminergic or noradrenergic nerve terminals which are not present in primary cultures of rat striatum.

Influence of PLA2-PG system on purine release and cAMP content in dissociated primary glial cultures from rat striatum

Purine release and prostaglandin (PG) outflow were simultaneously evaluated from untreated glial primary cultures of rat striatum, at rest and under field electrical stimulation. Purine release was also assayed from sister cultured cells in which a suitable pharmacological treatment with 1 x 10(-6) M dexamethasone or 1 x 10(-4) M indomethacin had produced a complete inhibition of the phospholipase A2-prostaglandin (PLA2-PG) system. Purine release from untreated cells seems to be regulated by specific receptor sites for released adenosine (Ado); A1 receptors exert an inhibitory control on purine release while A2 receptors facilitate it. PG release appears to be related to A1-mediated Ado activity, since culture treatment with 1 x 10(-10) M 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or 1 x 10(-4) M N-ethylmaleimide (NEM), A1 receptor inhibitory agents able to increase purine release, induced a significant reduction of the evoked PG outflow. Purine amount, released from glial cells with inhibited PLA2-PG system, was remarkably greater than that one assayed from control cultured cells. In so treated cultures, no additive effect, NEM-induced, was detected, while the addition of a mixture of PGs partially reduced the increased purine outflow. An electrically evoked cAMP accumulation, significantly greater than that found in controls, was even detected in cultured cells with inhibited PLA2-PG system. Since 10 micrograms/ml adenosine deaminase (ADA) reduced while DPCPX enhanced the evoked cAMP accumulation, it seems partially due to released Ado and accounts for a prevalent A2-stimulating rather than an A1-inhibitory control on adenylate cyclase activity. Thus, in cultured glial cells, the PLA2-PG system, likely linked to A1 receptor sites, concurs to control purine release and seems to affect less directly cAMP accumulation.