Primary Mesencephalic Cell Culture Research Articles

Calcium-dependent activator protein for secretion 2 is involved in dopamine release in mouse midbrain neurons.

The Ca2+-dependent activator protein for secretion (CAPS/CADPS) family protein facilitates catecholamine release through the dense-core vesicle exocytosis in model neuroendocrine cell lines. However, it remains unclear if it induces dopamine release in the central neurons. This study aimed to examine the expression and function of CADPS2, one of the two CADPS paralogs, in dopamine neurons of the mouse midbrain. This study shows that CADPS2 was expressed in tyrosine hydroxylase and the vesicular monoamine transporter 2 (VMAT2)-positive dopaminergic neurons of the midbrain samples and primary mesencephalic cell cultures. Subcellular fractions rich in dopamine were collected using immunoaffinity for CADPS2 from midbrain protein extracts. Cell imaging using fluorescent false neurotransmitter FFN511 as a substrate for VMAT2 showed decreased activity-dependent dopamine release in Cadps2-deficient cultures, compared to that in wild-type cultures. These results suggest that CADPS2 is involved in dopamine release from the central neurons, indicating its involvement in the central dopamine pathway.

Neuroprotective Effects of Delta-9-Tetrahydrocannabinol against FeSO4- and H2O2-Induced Cell Damage on Dopaminergic Neurons in Primary Mesencephalic Cell Culture

AbstractDelta-9-Tetrahydrocannabinol and other phytocannabinoids have been previously demonstrated to possess neuroprotective effects in murine mesencephalic cell culture models of Parkinson’s disease, in which increased levels of superoxide radicals led to the loss of dopaminergic neurons. In these models, delta-9-tetrahydrocannabinol did not scavenge these radicals but displayed antioxidative capacity by increasing glutathione levels. Based on these findings, in the present study, we investigated whether the neuroprotective effect of delta-9-tetrahydrocannabinol can also be detected in FeSO4- and H2O2-stressed cells. Mesencephalic cultures were concomitantly treated with FeSO4 (350 μM) or H2O2 (150 μM) and delta-9-tetrahydrocannabinol (0.01, 0.1, 1, 10 μM) on the 12th days in vitro for 48 h. On the 14th DIV, dopaminergic neurons were stained immunocytochemically by tyrosine hydroxylase, and fluorescently using crystal violet, Hoechst 33342, and JC-1. FeSO4 and H2O2 significantly reduced the number of dopaminergic neurons by 33 and 36%, respectively, and adversely affected the morphology of surviving neurons. Moreover, FeSO4, but not H2O2, significantly decreased the fluorescence intensity of crystal violet and Hoechst 33342, and reduced the red/green ratio of JC-1. Co-treatment with delta-9-tetrahydrocannabinol at the concentrations 0.01 and 0.1 μM significantly rescued dopaminergic neurons in FeSO4 and H2O2-treated cultures by 16 and 30%, respectively. delta-9-Tetrahydrocannabinol treatment also led to a higher fluorescence intensity of crystal violet and Hoechst 33342, and increased the red/green fluorescence ratio of JC-1 when concomitantly administered with FeSO4 but not H2O2. To conclude, delta-9-tetrahydrocannabinol rescues dopaminergic neurons against FeSO4- and H2O2-induced neurotoxicity. Using fluorescence dyes, this effect seems to be mediated partially by restoring mitochondrial integrity and decreasing cell death, particularly in FeSO4-treated cultures.

Autophagy Inhibitor 3-Methyladenine could not Modulate Rotenone Neurotoxicity in Primary Mesencephalic Cell Culture

Autophagy Inhibitor 3-Methyladenine could not Modulate Rotenone Neurotoxicity in Primary Mesencephalic Cell Culture

Neurotoxic effects of acrylamide on dopaminergic neurons in primary mesencephalic cell culture

Exposure to acrylamide is increasing worldwide as a result of its heavy use in industry and formation in carbohydrate-rich food cooked at high temperature. Despite its neurotoxicity, no studies have shown its toxic effects on dopaminergic neurons yet. Therefore, the current study was carried out to show whether acrylamide adversely affects primary cultured dopaminergic neurons. Acrylamide (0.001, 0.01, 0.1, 1, 2 mM) was added to two different groups of primary mesencephalic cell cultures on the 9th day in vitro for 24 and 48 h, respectively. Moreover, a group of cultures was treated with lower concentrations of acrylamide (0.01, 0.05, 0.1, 0.5 mM) on the 6th day in vitro for 5 consecutive days to investigate its long-term effects on dopaminergic neurons. Following each treatment, culture media were obtained for measuring lactate dehydrogenase, and cultured cells were stained immunocytochemically against tyrosine hydroxylase and neuronal nuclear antigens. Treatment of cultures with acrylamide for 48 h significantly reduced the number of dopaminergic neurons, adversely altered the morphology of the surviving neurons and increased levels of lactate dehydrogenase in the culture media. Similar treatment of cultures with acrylamide also resulted in lower numbers of total neuronal cells as shown by a reduced expression of the neuronal nuclear antigen. Prolonged treatment of cultures with lower concentrations of acrylamide slightly reduced the survival of dopaminergic neurons but increased the release of lactate dehydrogenase into the culture media as well. The current study shows, for the first time, neurotoxicity of acrylamide on dopaminergic neurons in the primary mesencephalic cell culture.

Long-term neurotoxic effects of domoic acid on primary dopaminergic neurons

Domoic acid, an excitatory neurotoxin produced by certain algae, reaches the food chain through accumulation in some sea organisms. To investigate its long-term neurotoxicity on dopaminergic neurons, prepared primary mesencephalic cell cultures were exposed to different concentrations of domoic acid (0.1, 1, 10, 100 μM) on the 8th day in vitro (DIV) for 4 days. On the 12th DIV, culture media were collected for measurement of lactate dehydrogenase and cultured cells were subjected to immunohistochemistry against tyrosine hydroxylase, neuronal nuclear antigen and glial fibrillary acidic protein, and fluorescence staining using H2DCFDA, JC-1 and Hoechst 33342 dyes. Moreover, roles of AMPA/KA and NMDA receptors in domoic acid neurotoxicity were also investigated. Domoic acid significantly decreased the number of dopaminergic neurons and adversely affected their morphology, and slightly reduced the expression of neuronal nuclear antigen and glial fibrillary acidic protein. Co-treatment of cultures with domoic acid and the AMPA/KA or NMDA receptor antagonists NBQX and MK-801 rescued significant number of dopaminergic neurons. Domoic acid significantly decreased red:green fluorescence ratio of JC-1 and did not affect production of reactive oxygen species and apoptotic cell death. In conclusions, the present study reveals that long-term treatment of primary mesencephalic cell culture with domoic acid significantly destroyed dopaminergic neurons. This effect appears to be attributed to activation of AMPA/KA and NMDA receptors and mitochondrial damage.

Neurotoxic effects of domoic acid on dopaminergic neurons in primary mesencephalic cell culture.

Domoic acid is a potent marine neurotoxin produced by certain species of the diatom genus Pseudonitzschia. To our knowledge, there are no studies that have investigated neurotoxic effects of domoic acid on dopaminergic neurons. Accordingly, the present study was carried out to investigate the potential neurotoxic effects of domoic acid on dopaminergic neurons in primary mesencephalic cell culture. Cultures prepared from embryonic mouse mesencephala (total of 250 embryos) were treated with different concentrations of domoic acid (0.1, 1, 10, 100 µM) on the 10th DIV for 48 h. On the 12th DIV, culture media were used for measurement of lactate dehydrogenase and cultured cells were subjected to immunostaining for tyrosine hydroxylase, neuronal nuclear antigen and glial fibrillary acidic protein, and fluorescence staining using H2DCFDA, JC-1 and DAPI stains. Moreover, roles of AMPA/KA and NMDA receptors in domoic acid neurotoxicity were also investigated. Domoic acid significantly decreased the number of dopaminergic neurons, decreased the expression of neuronal nuclear antigen and slightly affected astrocyte populations, and increased the release of lactate dehydrogenase into the culture media. AMPA/KA receptor antagonist NBQX but not NMDA receptor antagonist MK-801 significantly inhibited the neurotoxic effect of domoic acid on dopaminergic neurons. H<SUB>2</SUB>DCFDA, JC-1 and DAPI fluorescence staining, respectively, revealed that DomA slightly raised ROS production, and significantly decreased mitochondrial membrane potential and increased apoptotic cell death of cultured cells. The current study presents for the first time the neurotoxic effects of domoic acid on dopaminergic neurons and this effect appears to be attributed to activation of AMPA/KA receptors on dopaminergic neurons.

Rapamycin protects dopaminergic neurons against rotenone-induced cell death in primary mesencephalic cell culture.

Parkinson's disease is the most common movement disorder, characterized by a progressive and extensive loss of dopaminergic neurons in the substantia nigra pars compacta and their terminals in the striatum. So far, only symptomatic treatment is available, and no cure or disease-modifying drugs exist. The present study was designed to investigate the neuroprotective effect of rapamycin, an autophagy inducer, on dopaminergic neurons against rotenone-induced cell death in primary mesencephalic cell culture. Primary mesencephalic cell cultures were prepared from embryonic mouse mesencephala (OFI/SPF, Vienna, Austria) at gestation day 14. Four sets of cultures were treated as follows: one was run as an untreated control, a second one was treated with 20 nM rotenone on the 10th day in vitro (DIV) for 48 h, a third one was co-treated with 20 nM rotenone and rapamycin (1, 10, 100, 1000 nM) on the 10th DIV for 48 h, and a fourth one was treated with rapamycin alone (1, 10, 100, 1000 nM) on the 10th DIV for 48 h. On the 12th DIV, cultures were subjected to immunohistochemistry against tyrosine hydroxylase and to fluorescence staining using LysoTracker Deep Red, JC-1 and DAPI stains. Exposure of such cultures to 20 nM rotenone on the 10th DIV for 48 h reduced the number of dopaminergic neurons by 41% and increased the release of lactate dehydrogenase (LDH) by 178% above untreated controls. Rapamycin (1, 10, 100, 1000 nM) added together with rotenone from the 10th to 12th DIV spared dopaminergic neurons by 17% and reduced the release of LDH by 64% at the concentration of 100 nM compared to rotenone-treated cultures. Activation of an autophagic process by rapamycin was demonstrated by LysoTracker Deep Red fluorescent dye, as indicated by a shift to increased red fluorescence. Rapamycin also significantly elevated the mitochondrial membrane potential (Δψm), as shown by an increase of the red:green fluorescence ratio of JC-1. Increased apoptotic cell death due to rotenone was lowered by rapamycin, as shown by the blue-fluorescent DAPI nucleic acid stain. Our study indicates for the first time that rapamycin, known as an autophagy inducer, protected dopaminergic neurons against rotenone-induced cell death in primary mesencephalic cell culture.

Cabergoline protects dopaminergic neurons against rotenone-induced cell death in primary mesencephalic cell culture.

In the present study, primary mesencephalic cell cultures prepared from embryonic mouse mesencephala were used to investigate the neuroprotective effect of cabergoline, an ergoline D2 receptor agonist, against the pesticide and neurotoxin rotenone relevant to Parkinson disease (PD). Treatment of cultures with cabergoline alone significantly increased the number of tyrosine hydroxylase immunoreactive (THir) neurons and reduced the release of lactate dehydrogenase (LDH) into the culture medium compared to untreated controls. Against rotenone toxicity, cabergoline significantly rescued degenerating THir neurons, reduced the release of LDH into the culture medium and improved the morphology of surviving THir neurons. The neuroprotective effects afforded by cabergoline were independent of dopaminergic stimulation as blocking of dopamine receptors by the dopamine receptor antagonist sulpiride did not prevent them. Furthermore, rotenone-induced formation of reactive oxygen species (ROS) was significantly reduced by cabergoline. Although cabergoline increased the glutathione (GSH) content in the culture, the protective effect for dopaminergic neurons seemed not to be predominantly mediated by increasing GSH, as depletion of GSH by L-buthionine-(S,R)-sulfoximine (BSO), a GSH biosynthesis inhibitor, did not prevent cabergoline-mediated neuroprotection of THir neurons in rotenone-treated cultures. Moreover, cabergoline significantly increased the ATP/protein ratio in primary mesencephalic cell cultures when added alone or prior to rotenone treatment. These results indicate a neuroprotective effect of cabergoline for dopaminergic neurons against rotenone toxicity. This effect was independent of dopamine receptor stimulation and was at least partially mediated by reducing ROS production and increasing the ATP/protein ratio.

Neuroprotective effect of rotigotine against complex I inhibitors, MPP⁺ and rotenone, in primary mesencephalic cell culture.

Dopamine agonists are suggested to be more efficacious in treating Parkinson's disease (PD) as they have neuroprotective properties in addition to their receptor-related actions. The present study was designed to investigate the neuroprotective effects of rotigotine, a D3/D2/D1 dopamine receptor agonist, against the two powerful complex I inhibitors, 1-methyl-4-phenylpyridinium (MPP+) and rotenone, in primary mesencephalic cell culture relevant to PD. Primary mesencephalic cell cultures were prepared from embryonic mouse mesencephala at gestation day 14. Three sets of cultures were treated with rotigotine alone, rotigotine and MPP⁺, and rotigotine and rotenone to investigate the effect of rotigotine on the survival of dopaminergic neurons against age-, MPP⁺- and rotenone-induced cell death. At the end of each treatment, cultures were fixed and stained immunohistochemically against tyrosine hydroxylase (TH). The effect of rotigotine against rotenone-induced reactive oxygen species (ROS) production was measured using CH-H2DCFDA fluorescence dye. Rotigotine alone did not influence the survival of tyrosine hydroxylase immunoreactive (THir) neurons except at 10 µM, it significantly decreased the number of THir neurons by 40% compared to untreated controls. Treatment of cultures with 0.01 µM rotigotine rescued 10% of THir neurons against MPP⁺-induced cell death. Rotigotine was also found to significantly rescue 20% of THir neurons at 0.01 µM of rotenone-treated cultures. Using of CH-H2DCFDA fluorescence dye, it was found that rotigotine significantly attenuated ROS production compared to rotenone-treated cultures. Rotigotine provides minor protection against MPP⁺ and rescues a significant number of THir neurons against rotenone in primary mesencephalic cell cultures relevant to PD.

Nitric oxide/soluble guanylyl cyclase signaling mediates depolarization-induced protection of rat mesencephalic dopaminergic neurons from MPP+ cytotoxicity

Neuronal electrical activity has been known to affect the viability of neurons in the central nervous system. Here we show that long-lasting membrane depolarization induced by elevated extracellular K+ recruits nitric oxide (NO)/soluble guanylyl cyclase/protein kinase G signaling pathway, induces 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP)-mediated protein S-guanylation, and confers dopaminergic neuroprotection. Treatment of primary mesencephalic cell cultures with 1-methyl-4-phenylpyridinium (MPP+) for 72h decreased the number of dopaminergic neurons, whereas the cell loss was markedly inhibited by elevated extracellular concentration of K+ (+40mM). The neuroprotective effect of elevated extracellular K+ was significantly attenuated by tetrodotoxin (a Na+ channel blocker), amlodipine (a voltage-dependent Ca2+ channel blocker), Nω-nitro-l-arginine methyl ester (l-NAME) (a nitric oxide synthase inhibitor), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (a soluble guanylyl cyclase inhibitor), and KT5823 or Rp-8-bromo-β-phenyl-1,N2-ethenoguanosine 3′,5′-cyclic monophosphorothioate (Rp-8-Br-PET-cGMPS) (protein kinase G inhibitors). Elevated extracellular K+ increased 8-nitro-cGMP production resulting in the induction of protein S-guanylation in cells in mesencephalic cultures including dopaminergic neurons. In addition, exogenous application of 8-nitro-cGMP protected dopaminergic neurons from MPP+ cytotoxicity, which was prevented by zinc protoporphyrin IX, an inhibitor of heme oxygenase-1 (HO-1). Zinc protoporphyrin IX also inhibited the neuroprotective effect of elevated extracellular K+. On the other hand, KT5823 or Rp-8-Br-PET-cGMPS did not inhibit the induction of HO-1 protein expression by 8-nitro-cGMP, although these protein kinase G inhibitors abrogated the neuroprotective effect of 8-nitro-cGMP. These results suggest that protein S-guanylation (leading to HO-1 induction) as well as canonical protein kinase G signaling pathway plays an important role in NO-mediated, activity-dependent dopaminergic neuroprotection.

HIF prolyl hydroxylase inhibition increases cell viability and potentiates dopamine release in dopaminergic cells

Hypoxia-inducible factor (HIF) controls the expression of genes that adapts the cellular condition to accommodate oxidative stress. The potential beneficial effect of HIF up-regulation in ischemia has recently gained interest substantiated by the known HIF-regulation of erythropoietin and other hypoxia accommodating genes. So far the perspectives for HIF up-regulation has been focused on anemia and ischemia related diseases but little information is available about the relevance of HIF biology for neurodegenerative disease like Parkinson's disease. We therefore sought out to characterize the effect of HIF-up-regulation on survival and dopamine homeostasis in dopaminergic cells. We used a low molecular weight HIF prolyl hydroxylase (HPH) inhibitor and lentiviral based shRNA knockdown of HPH subtypes as molecular tools to increase HIF protein level and downstream HIF-regulated genes. We show that HIF induction results in protection against oxidative stress in cellular models based on PC12 cells and LUHMES cells. In addition, HPH inhibition elevates tyrosine hydroxylase expression and activity, which causes increased dopamine synthesis and release in both PC12 cells and a primary rat ventral mesencephalic cell culture. All together these findings suggest that prolyl hydroxylases may represent novel targets for therapeutic intervention in disorders characterized by dopamine homeostasis dysregulation like Parkinson's disease.

Direct Visualization of Internalization and Intracellular Trafficking of Dopamine-Releasing Protein-36aa

Dopamine-releasing protein (DARP) is a potent regulator of dopamine (DA) release known to be involved in the development of rat catecholaminergic systems. In the present study, we examined the internalization and transport of DARP-36aa, a synthetic peptide derived from the N-terminal sequence of DARP, in rat C6 glioma cells. A colloidal gold DARP-36aa conjugate (DARP-36aa: AU) and biotinylated DARP-36aa were employed to visualize internalization and intracellular transport of DARP-36aa. Electron microscopy demonstrated that DARP-36aa: AU was rapidly incorporated into C6 glioma cells. Internalization via clathrin-coated pits and vesicles was clearly observed followed by transport and sorting of DARP-36aa: AU into multivesicular bodies, tubulo-vesicular endosomes, and lysosomes. Internalization of DARP-36aa: AU was also examined in primary mesencephalic cell cultures where a similar pattern of internalization and transport via clathrin-coated pits and vesicles was observed. Fluorescence microscopy using a biotinylated DARP-36aa/avidin-rhodamine conjugate revealed that DARP-36aa is diffusely distributed on the plasmalemma prior to internalization at 4°C. Following a 30-min incubation at 37°C DARP-36aa was concentrated in the cytosol, particularly in areas surrounding cellular projections and the perikaryon. Immunocytochemical studies employing biotinylated DARP-36aa and an anti-clathrin heavy chain antibody demonstrated that DARP-36aa and clathrin colocalize during DARP-36aa internalization. We also observed a marked increase in tyrosine phosphorylation of a 45-kD protein in response to DARP-33a stimulation in C6 glioma cells. Genistein, a specific tyrosine kinase inhibitor, significantly inhibited DARP-36aa: AU internalization and transport in C6 glioma cells. These findings suggest that tyrosine kinase activity may result in DARP-36aa receptor-mediated endocytosis.

Microglial Activation and Dopaminergic Cell Injury: AnIn VitroModel Relevant to Parkinson's Disease

Microglial activation and oxidative stress are significant components of the pathology of Parkinson's disease (PD), but their exact contributions to disease pathogenesis are unclear. We have developed an in vitro model of nigral injury, in which lipopolysaccharide-induced microglial activation leads to injury of a dopaminergic cell line (MES 23.5 cells) and dopaminergic neurons in primary mesencephalic cell cultures. The microglia are also activated by PD IgGs in the presence of low-dose dopa-quinone- or H(2)O(2)-modified dopaminergic cell membranes but not cholinergic cell membranes. The activation requires the microglial FCgammaR receptor as demonstrated by the lack of activation with PD IgG Fab fragments or microglia from FCgammaR-/- mice. Although microglial activation results in the release of several cytokines and reactive oxygen species, only nitric oxide and H(2)O(2) appear to mediate the microglia-induced dopaminergic cell injury. These studies suggest a significant role for microglia in dopaminergic cell injury and provide a mechanism whereby immune/inflammatory reactions in PD could target oxidative injury relatively specifically to dopaminergic cells.

Is Bax a mitochondrial mediator in apoptotic death of dopaminergic neurons in Parkinson's disease?

Bax is a proapoptotic member of the Bcl-2 family of proteins. It is believed to exert its action primarily by facilitating the release of cytochrome c from the mitochondrial intermembrane space into the cytosol, leading to caspase activation and cell death. Because alterations in mitochondrial respiratory function, caspase activation and cell death with morphologic features compatible with apoptosis have been observed post mortem in the brain of patients with Parkinson's disease, we tried to clarify the potential role of Bax in this process in an immunohistochemical study on normal and Parkinson's disease post-mortem brain and primary mesencephalic cell cultures treated with MPP(+). We found that Bax is expressed ubiquitously by dopaminergic (DA) neurons in post-mortem brain of normal and Parkinson's disease subjects as well as in vitro. Using an antibody to Bax inserted into the outer mitochondrial membrane as an index of Bax activation, no significant differences were observed between control and Parkinson's disease subjects, regardless of the mesencephalic subregion analysed. However, in Parkinson's disease subjects, the percentage of Bax-positive melanized SNpc neurons containing Lewy bodies, suggestive of DA neuronal suffering, was significantly higher than the overall percentage of Bax-positive neurons among melanized neurons. Furthermore, all melanized SNpc neurons in Parkinson's disease subjects with activated caspase-3 were also immunoreactive for Bax, suggesting that Bax anchored in the outer mitochondrial membrane of melanized SNpc neurons showing signs of neuronal suffering or apoptosis is increased compared with DA neurons that are apparently unaltered. Surprisingly, MPP(+) treatment of tyrosine hydroxylase (TH)-positive neurons in primary mesencephalic cultures did not cause redistribution of Bax, although cytochrome c was released from the mitochondria and nuclear condensation/fragmentation was induced. Taken together, these findings suggest that in the human pathology, Bax may be a cofactor in caspase activation, but our in vitro data fail to indicate a central role for Bax in apoptotic death of DA neurons in an experimental Parkinson's disease paradigm.

Cellular localization of dopamine-releasing protein (DARP) in rat C6 glioma and primary mesencephalic cell cultures

Dopamine-releasing protein (DARP) is a multisubunit protein shown to have dramatic effects on development, recovery, and function of the rat catecholaminergic (CA) system. This study details efforts to determine if glial cells are responsible for the production of DARP in the central nervous system (CNS). Enzyme-linked immunosorbent assays (ELISA), Western blotting, and immunocytochemical techniques were employed to measure DARP levels and identify DARP immunoreactive proteins in rat C6 glioma cells and medium, respectively. ELISA analysis of serum-free C6 culture media revealed a maximal concentration of DARP by culture day 1. However, ELISA analysis of C6 cultures grown in F-12K/serum medium revealed that maximal levels of DARP were detected on culture day 6 with a 108% increase in DARP immunoreactivity from culture day 1. These values were determined using a polyclonal antibody generated against DARP-36aa (anti-DARP-36aa), a synthetic peptide with dopamine (DA) releasing activity, and anti-DARP B9–B10, a monoclonal antibody generated against partially purified DARP. Western blot analysis revealed that anti-DARP B9–B10 recognized proteins of approximately 60, 50, and 45 kDa in C6 cell homogenates while anti-DARP-36aa had immunoreactivity with the 60-kDa protein alone. Immunocytochemical studies demonstrated that anti-DARP-36aa and anti-DARP B9–B10 had strong immunoreactivity with proteins throughout the cytosol and in several processes of C6 cells. These results reveal that DARP is detected in glioma cells and secreted in a time-dependent fashion during culture. Primary rat mesencephalic cultures were also examined using immunocytochemistry. Incubation with DARP antibodies and antisera against glial fibrillary acidic protein (GFAP) revealed that DARP and GFAP immunoreactivity co-localized in primary mesencephalic cultures. However, the majority of DARP immunoreactivity was localized to cells without GFAP staining. These findings reveal that DARP is detected in astrocytes although the majority of DARP immunoreactivity is found in non-astrocyte type cells.

Hypoxia during early developmental period induces long-term changes in the dopamine content and release in a mesencephalic cell culture

The present study was conducted to elucidate the long-term effects of exposure to hypoxia of dopaminergic neurons during the early developmental period. Primary mesencephalic cell cultures prepared from fetal rats and containing 0.5–2% of dopaminergic neurons were exposed to hypoxia between in vitro days 1 and 6, the putative critical developmental period. Changes in the content, release and uptake of dopamine were found to depend on the degree of hypoxia and on the duration of exposure. Following moderate hypoxia (7 h, 5% O 2) on two consecutive days between in vitro days 1 and 3, the cultures showed a small increase in the dopamine levels, by 16%. After severe hypoxia (0% O 2/95% N 2 for 24 h), during the same time window, the cellular dopamine content was elevated by 100%. Moreover, severe hypoxia produced long-lasting modulations of the dopaminergic system. On in vitro day 14, cells exhibited increased levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid (by 34% and 55%, respectively), and elevations of both the spontaneous and potassium-stimulated dopamine release by 70%. The dopamine transport and metabolism of cells exposed to hypoxia between in vitro days 4 and 6 remained unchanged with regard to long-term effects. The present study provides strong evidence for the induction of long-term changes in dopaminergic cells due to hypoxia during the critical developmental period in mesencephalic culture. The developmental period capable of inducing long-lasting changes in dopamine metabolism is restricted to in vitro days 1–3.

Quantitation of dopamine D 2 receptor mRNA in a mesencephalic cell culture using a nonradioactive competitive reverse transcription polymerase chain reaction method

Studies on gene expression during differentiation and maturation processes have to cope with determinations of extremely low steady state levels of specific mRNA. Using the experimental model of dopamine D 2 receptor (D 2R) expression in a primary mesencephalic cell culture we worked out a quantitative reverse transcription polymerase chain reaction method which allows to analyze and quantify mRNA levels of cells present in a few wells of the culture. The method uses an internal cRNA standard which shares both primer binding sites and PCR product length with the target sequence. The amplicons are quantitated in microplates by hybridization with immobilized capture probes that allow for the distinction of internal standard and target sequences followed by the chemiluminescent detection of hybridized DNA. Applying this method the levels of D 2 receptor mRNA of the mesencephalic cell culture on day in vitro 1 amounted to about 250 fg/μg RNA and increased to about 1200 fg/μg RNA on day in vitro 13–15.

65 Detection of presenilin I protein in the brain and primary mesencephalic cell cultures

65 Detection of presenilin I protein in the brain and primary mesencephalic cell cultures

Measurement of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in cultured rat mesencephalic neurons by high-performance liquid chromatography with electrochemical detection

An HPLC method with electrochemical detection for the simultaneous measurement of serotonin (5-hydroxytryptamine) and 5-hydroxyindoleacetic acid in primary mesencephalic cell culture is described. The serotonin and 5-hydroxyindoleacetic acid cell content was measured on different days of growth in vitro; after twelve days in culture the amounts of serotonin and 5-hydroxyindoleacetic acid detected were 916.0 ± 70.2 and 215.8 ± 15.5 pg per well, respectively. The heterogeneity of neurons in our cultures and their capacity to take up serotonin were assessed by measuring the amounts of exogenous serotonin taken up in the presence of different monoamine uptake inhibitors. This method, sensitive and reliable, can represent a valid alternative to the use of labelled compounds.