Enhanced proliferation and dopaminergic differentiation of ventral mesencephalic precursor cells by synergistic effect of FGF2 and reduced oxygen tension.

Effective numerical expansion of dopaminergic precursors might overcome the limited availability of transplantable cells in replacement strategies for Parkinson's disease. Here we investigated the effect of fibroblast growth factor-2 (FGF2) and FGF8 on expansion and dopaminergic differentiation of rat embryonic ventral mesencephalic neuroblasts cultured at high (20%) and low (3%) oxygen tension. More cells incorporated bromodeoxyuridine in cultures expanded at low as compared to high oxygen tension, and after 6 days of differentiation there were significantly more neuronal cells in low than in high oxygen cultures. Low oxygen during FGF2-mediated expansion resulted also in a significant increase in tyrosine hydroxylase-immunoreactive (TH-ir) dopaminergic neurons as compared to high oxygen tension, but no corresponding effect was observed for dopamine release into the culture medium. However, switching FGF2-expanded cultures from low to high oxygen tension during the last two days of differentiation significantly enhanced dopamine release and intracellular dopamine levels as compared to all other treatment groups. In addition, the short-term exposure to high oxygen enhanced in situ assessed TH enzyme activity, which may explain the elevated dopamine levels. Our findings demonstrate that modulation of oxygen tension is a recognizable factor for in vitro expansion and dopaminergic differentiation of rat embryonic midbrain precursor cells.

A human stem cell-based model for identifying adverse effects of organic and inorganic chemicals on the developing nervous system.

The aim of our study was to investigate whether a human neural stem cell line derived from umbilical cord blood (HUCB-NSC) can serve as a reliable test model for developmental neurotoxicity (DNT). We assessed the sensitivity of HUCB-NSCs at different developmental stages to a panel of neurotoxic (sodium tellurite, methylmercury chloride, cadmium chloride, chlorpyrifos, and L-glutamate) and non-neurotoxic (acetaminophen, theophylline, and D-glutamate) compounds. In addition, we investigated the effect of some compounds on key neurodevelopmental processes like cell proliferation, apoptotic cell death, and neuronal and glial differentiation. Less differentiated HUCB-NSCs were generally more sensitive to neurotoxicants, with the notable exception of L-glutamate, which showed a higher toxicity to later stages. The relative potencies of the compounds were: cadmium chloride > methylmercury chloride >> chlorpyrifos >> L-glutamate. Fifty nanomolar methylmercury chloride (MeHgCl) inhibited proliferation and induced apoptosis in early-stage cells. At the differentiated stage, 1 muM MeHgCl induced selective loss of S100 beta-expressing astrocytic cells. One millimolar L-glutamate did not influence the early stages of HUCB-NSC development, but it affected late stages of neuronal differentiation. A valuable system for in vitro DNT assessment should be able to discriminate between neurotoxic and non-neurotoxic compounds and show different susceptibilities to chemicals according to developmental stage and cell lineage. Although not exhaustive, this work shows that the HUCB-NSC model fulfils these criteria and may serve as a human in vitro model for DNT priority setting.

Enhanced dopaminergic differentiation of human neural stem cells by synergistic effect of Bcl-xL and reduced oxygen tension.

Neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. Here we investigated the effect of the anti-apoptotic protein Bcl-x(L) and oxygen tension on dopaminergic differentiation and survival of a human ventral mesencephalic stem cell line (hVM1). hVM1 cells and a Bcl-x(L) over-expressing subline (hVMbcl-x(L)) were differentiated by sequential treatment with fibroblast growth factor-8, forskolin, sonic hedgehog, and glial cell line-derived neurotrophic factor. After 10 days at 20% oxygen, hVMbcl-x(L) cultures contained proportionally more tyrosine hydroxylase(TH)-positive cells than hVM1 control cultures. This difference was significantly potentiated from 11 +/- 0.8% to 17.2 +/- 0.2% of total cells when the oxygen tension was lowered to 3%. Immunocytochemistry and Q-PCR-analysis revealed expression of several dopaminergic markers besides of TH just as dopamine was detected in the culture medium by HPLC analysis. Although Bcl-x(L)-over-expression reduced cell death in the cultures, it did not alter the relative content of GABAergic, neurons, while the content of astroglial cells was reduced in hVMbcl-x(L) cell cultures compared with control. We conclude that Bcl-x(L) and lowered oxygen tension act in concert to enhance dopaminergic differentiation and survival of human neural stem cells.

Dopaminergic differentiation of human neural stem cells mediated by co-cultured rat striatal brain slices.

Properly committed neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. To establish a setting for identification of secreted neural compounds promoting dopaminergic differentiation, we co-cultured cells from a human neural forebrain-derived stem cell line (hNS1) with rat striatal brain slices. In brief, coronal slices of neonatal rat striatum were cultured on semiporous membrane inserts placed in six-well trays overlying monolayers of hNS1 cells. After 12 days of co-culture, large numbers of tyrosine hydroxylase (TH)-immunoreactive, catecholaminergic cells could be found underneath individual striatal slices. Cell counting revealed that up to 25.3% (average 16.1%) of the total number of cells in these areas were TH-positive, contrasting a few TH-positive cells (<1%) in non-induced areas. The presence of dopamine in the conditioned culture medium was confirmed by HPLC analysis. Interestingly, not all striatal slice cultures induced TH-expression in underlying hNS1 cells. Common to TH-inductive cultures was, however, the presence of degenerating, necrotic areas, suggesting that factors released during striatal degeneration were responsible for the dopaminergic induction of the hNS1 cells. Ongoing experiments aim to identify such factors by comparing protein profiles of media conditioned by degenerating (necrotic) versus healthy striatal slice cultures.

Functional effect of FGF2- and FGF8-expanded ventral mesencephalic precursor cells in a rat model of Parkinson's disease.

Ventral mesencephalic (VM) precursor cells are of interest in the search for transplantable dopaminergic neurons for cell therapy in Parkinson's disease (PD). In the present study we investigated the survival and functional capacity of in vitro expanded, primary VM precursor cells after intrastriatal grafting to a rat model of PD. Embryonic day 12 rat VM tissue was mechanically dissociated and cultured for 4 or 8 days in vitro (DIV) in the presence of FGF2 (20 ng/ml), FGF8 (20 ng/ml) or without mitogens (control). Cells were thereafter differentiated for 6 DIV by mitogen withdrawal and addition of serum. After differentiation, significantly more tyrosine hydroxylase-immunoreactive (TH-ir), dopamine-producing neurons were found in FGF2- and FGF8-expanded cultures compared to controls. Moreover, expansion for 4 DIV resulted in significantly more TH-ir cells than expansion for 8 DIV both for FGF2 (2.4 fold; P<0.001) and FGF8 (3.8 fold; P<0.001) treated cultures. The functional potential of the expanded cells (4 DIV) was examined after grafting into striatum of aged 6-hydroxydopamine-lesioned rats. Amphetamine-induced rotations performed 3, 6 and 9 weeks postgrafting revealed that grafts of FGF2-expanded cells induced a significantly faster and better functional recovery than grafts of FGF8-expanded cells or control cells (P<0.05 for both). Grafts of FGF2-expanded cells also contained significantly more TH-ir cells than grafts of FGF8-expanded cells (P<0.05) or control cells (P<0.01). In conclusion, FGF2-mediated pregrafting expansion of primary VM precursor cells considerably improves dopaminergic cell survival and functional restoration in a rat model of PD.

Endonuclease G expression in thalamic reticular nucleus after global cerebral ischemia.

Endonuclease G is a mitochondrial enzyme, known to be translocated to the nucleus after transient focal cerebral ischemia and contribute to DNA degradation. After global cerebral ischemia, delayed cell death is observed in the thalamic reticular nucleus but the mechanisms involved are not well described. The purpose of this study was to identify if Endonuclease G was expressed in the cell nucleus of parvalbumin(+) GABA'ergic neurons in relation to cell death after global cerebral ischemia in the thalamic reticular nucleus. The cell death in male Wister rats were studied from 6 h until 4 days after global cerebral ischemia induced by transient 2-vessel carotid occlusion with hypotension for 15 min. Hematoxylin-eosin staining and immunohistochemistry for Endonuclease G, Parvalbumin and Glial fibrillary acidic protein was performed after the ischemic insult. Eosinophilic neurons and vacuolization of the cytoplasm in parvalbumin(+) neurons were observed 2 days after ischemia. Endonuclease G immunoreactivity increased in the cytoplasm 12 h after ischemia and was translocated to the nucleus of parvalbumin(+) neurons after 24 h. In the nucleus of astroglia, Endonuclease G was expressed after 2 days with an apoptotic-like morphology and the number of Endonuclease G-expressing astroglia increased during the later time points. During the same period the number of parvalbumin(+) neurons decreased. In conclusion, this study has identified that Endonuclease G is translocated from the cytoplasm to the nucleus of neurons and expressed with apoptotic-like morphology in the nucleus of astroglia in the thalamic reticular nucleus after global cerebral ischemia.

The dentate mossy fibers: structural organization, development and plasticity.

Hippocampal mossy fibers are the axons of the dentate granule cells and project to hippocampal CA3 pyramidal cells and mossy cells of the dentate hilus (CA4) as well as a number of interneurons in the two areas. Besides their role in hippocampal function, studies of which are still evolving and taking interesting turns, the mossy fibers display a number of unique features with regard to axonal projections, terminal structures and synaptic contacts, development and variations among species and strains, as well as to normal occurring and lesion-induced plasticity and neural transplantation. These features are the topic of this review, which will use the mossy fiber system of the rat as basis and reference in its aim to provide an up-to-date, yet historically based guide to students in the field.

Neural tissue-spheres: a microexplant culture method for propagation of precursor cells from the rat forebrain subventricular zone.

By combining new and established protocols we have developed a procedure for isolation and propagation of neural precursor cells from the forebrain subventricular zone (SVZ) of newborn rats. Small tissue blocks of the SVZ were dissected and propagated en bloc as free-floating neural tissue-spheres (NTS) in EGF and FGF2 containing medium. The spheres were cut into quarters when passaged every 10-15th day, avoiding mechanical or enzymatic dissociation in order to minimize cellular trauma and preserve intercellular contacts. For analysis of regional differences within the forebrain SVZ, NTS were derived from three rostro-caudal levels of the lateral ventricles (anterior, intermediate and posterior) and propagated separately. Explants from all three levels produced proliferating NTS, but "anterior" NTS in general grew to smaller sizes than "intermediate" and "posterior" NTS. Posterior NTS moreover maintained their neurogenic potential throughout 77 days of propagation, while the ability of anterior NTS to generate neurons severely declined from day 40. The present procedure describes isolation and long-term expansion of forebrain SVZ tissue with potential preservation of the endogenous cellular content, thus allowing experimental studies of neural precursor cells and their niche.

Neuroprotective effects of the AMPA antagonist PNQX in oxygen-glucose deprivation in mouse hippocampal slice cultures and global cerebral ischemia in gerbils.

PNQX (9-methyl-amino-6-nitro-hexahydro-benzo(F)quinoxalinedione) is a selective AMPA antagonist with demonstrated neuroprotective effects in focal ischemia in rats. Here we report corresponding effects in mouse hippocampal slice cultures subjected to oxygen and glucose deprivation (OGD) and in transient global cerebral ischemia in gerbils. For in vitro studies, hippocampal slice cultures derived from 7-day-old mice and grown for 14 days, were submersed in oxygen-glucose deprived medium for 30 min and exposed to PNQX for 24 h, starting together with OGD, immediately after OGD, or 2 h after OGD. For comparison, other cultures were exposed to the NMDA antagonist MK-801 using the same protocol. Both PNQX and MK-801 displayed significant neuroprotective effects in all hippocampal subfields when present during and after OGD. When added just after OGD, only PNQX retained some neuroprotective effect. When added 2 h after OGD neither PNQX nor MK-801 had an effect. Transient global cerebral ischemia was induced in Mongolian gerbils by occlusion of both common carotid arteries for 4.5 min, with PNQX (10 mg/kg) being injected i.p. 30, 60 and 90 min after the insult. Subsequent analysis of brain sections stained for the neurodegeneration marker Fluoro-Jade B and immunostained for the astroglial marker glial fibrillary acidic protein revealed a significant PNQX-induced decrease in neuronal cell death and astroglial activation. We conclude that, PNQX provided neuroprotection against both global cerebral ischemia in gerbils in vivo and oxygen-glucose deprivation in mouse hippocampal slice cultures.

Long-term, repeated dose in vitro neurotoxicity of the glutamate receptor antagonist L-AP3, demonstrated in rat hippocampal slice cultures by using continuous propidium iodide incubation.

Most in vitro models are only used to assess short-term effects of test compounds. However, as demonstrated here, hippocampal slice cultures can be used for long-term studies. The test compound used was the metabotropic glutamate receptor antagonist, L(+)-2-amino-3-phosphonopropionic acid (L-AP3), which is known to be toxic in vivo after subchronic, but not acute, administration. Degenerative effects were monitored by measuring the cellular uptake of propidium iodide (PI; continuously present in the medium) and lactate dehydrogenase (LDH) leakage, and by using a panel of histological stains. Hippocampal slices, derived from 2-3 day old rats and grown for 3 weeks, were subsequently exposed for the next 3 weeks to 0, 10 or 100microM L-AP3, with PI (2microM) in the culture medium. Exposure to 100microM L-AP3 induced severe toxicity after 4-6 days, shown by massive PI uptake, LDH leakage, changes in MAP2 and GFAP immunostaining, and in Nissl and Timm staining. In contrast, 10microM L-AP3 did not induce detectable neuronal degeneration. Treatment with the NMDA receptor antagonist, MK-801, or the AMPA/KA receptor antagonist NBQX, together with 100microM L-AP3, reduced neurodegeneration down to close to control values. It is concluded that continuous incubation of hippocampal slice cultures with PI is technically feasible for use in studies of inducible neuronal degeneration over time.

The developmental expression of fluorescent proteins in organotypic hippocampal slice cultures from transgenic mice and its use in the determination of excitotoxic neurodegeneration.

Transgenic mice, expressing fluorescent proteins in neurons and glia, provide new opportunities for real-time microscopic monitoring of degenerative and regenerative structural changes. We have previously validated and compared a number of quantifiable markers for neuronal damage and cell death in organotypic brain slice cultures, such as cellular uptake of propidium iodide (PI), loss of microtubule-associated protein 2 (MAP2), Fluoro-Jade (FJ) cell staining, and the release of cytosolic lactate dehydrogenase (LDH). An important supplement to these markers would be data on corresponding morphological changes, as well as the opportunity to monitor reversible changes or long-term effects in the event of minor damage. As a first step, we present: a) the developmental expression in organotypic hippocampal brain slice cultures of transgenic fluorescent proteins, useful for the visualisation of neuronal subpopulations and astroglial cells; and b) examples of excitotoxic, glutamate receptor-induced degeneration of hippocampal CA1 pyramidal cells, with corresponding astroglial reactivity in such cultures. The slice cultures were set up according to standard techniques, by using one-week old pups from four transgenic mouse strains which express fluorescent proteins in their neurons and/or astroglial cells. From the time of explantation, and subsequently for up to nine weeks in culture, the transgenic neuronal fluorescence displayed the expected characteristics of a developmental, in vivo-like increase, including both the number and localisation of cells, as well as the intensity of fluorescence. At that stage and later, the transgenic fluorescence clearly permitted the visualisation of cell bodies, larger and smaller dendritic branches, spines and axons. In separate experiments, with a 24-hour exposure of matured sliced cultures to 100 microM of the glutamate agonist, N-methyl-D-aspartate (NMDA), we observed, by time-lapse recording, a gradual, but rapid loss of fluorescent CA1 pyramidal cells, accompanied by astrogliosis of transgene fluorescent astroglial cells. Based on these results, we consider that organotypic brain slice cultures from transgenic mice, with fluorescent neurons and glia, combined with detailed visualisation by time-lapse fluorescence microscopy, have great potential for investigating both major irreversible and minor reversible structural changes in neurons and glia, induced by neurotoxins and other neurodegenerative compounds and conditions.

Modulator effects of interleukin-1beta and tumor necrosis factor-alpha on AMPA-induced excitotoxicity in mouse organotypic hippocampal slice cultures.

The inflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha (TNF-alpha) have been identified as mediators of several forms of neurodegeneration in the brain. However, they can produce either deleterious or beneficial effects on neuronal function. We investigated the effects of these cytokines on neuronal death caused by exposure of mouse organotypic hippocampal slice cultures to toxic concentrations of AMPA. Either potentiation of excitotoxicity or neuroprotection was observed, depending on the concentration of the cytokines and the timing of exposure. A relatively high concentration of mouse recombinant TNF-alpha (10 ng/ml) enhanced excitotoxicity when the cultures were simultaneously exposed to AMPA and to this cytokine. Decreasing the concentration of TNF-alpha to 1 ng/ml resulted in neuroprotection against AMPA-induced neuronal death independently on the application protocol. By using TNF-alpha receptor (TNFR) knock-out mice, we demonstrated that the potentiation of AMPA-induced toxicity by TNF-alpha involves TNF receptor-1, whereas the neuroprotective effect is mediated by TNF receptor-2. AMPA exposure was associated with activation and proliferation of microglia as assessed by macrophage antigen-1 and bromodeoxyuridine immunohistochemistry, suggesting a functional recruitment of cytokine-producing cells at sites of neurodegeneration. Together, these findings are relevant for understanding the role of proinflammatory cytokines and microglia activation in acute and chronic excitotoxic conditions.

Toxic effects of lipid-mediated gene transfer in ventral mesencephalic explant cultures.

Adverse effects of cDNA and oligonucleotide delivery methods have not yet been systematically analyzed. We introduce a protocol to monitor toxic effects of two non-viral lipid-based gene delivery protocols using CNS primary tissue. Cell membrane damage was monitored by quantifying cellular uptake of propidium iodide and release of cytosolic lactate dehydrogenase to the culture medium. Using a liposomal transfection reagent, cell membrane damage was already seen 24 hr after transfection. Nestin-positive target cells, which were used as morphological correlate, were severely diminished in some areas of the cultures after liposomal transfection. In contrast, the non-liposomal transfection reagent revealed no signs of toxicity. This approach provides easily accessible information of transfection-associated toxicity and appears suitable for prescreening of transfection reagents.

Organotypic hippocampal slice cultures for studies of brain damage, neuroprotection and neurorepair.

Slices of developing brain tissue can be grown for several weeks as so-called organotypic slice cultures. Here we summarize and review studies using hippocampal slice cultures to investigate mechanisms and treatment strategies for the neurodegenerative disorders like stroke (cerebral ischemia), Alzheimer's disease (AD) and epilepsia. Studies of non-excitotoxic neurotoxic compounds and the experimental use of slice cultures in studies of HIV neurotoxicity, traumatic brain injury (TBI) and neurogenesis are included. For cerebral ischemia, experimental models with oxygen-glucose deprivation (OGD) and exposure to glutamate receptor agonists (excitotoxins) are reviewed. For epilepsia, focus is on induction of seizures with effects on neuronal loss, axonal sprouting and neurogenesis. For Alzheimer's disease, the review centers on the use of beta-amyloid (Abeta) in different models, while the section on repair is focused on neurogenesis and cell migration. The culturing techniques, set-up of models, and analytical tools, including markers for neurodegeneration, like the fluorescent dye propidium iodide (PI), are reviewed and discussed. Comparisons are made between hippocampal slice cultures and other in vitro models using dispersed cell cultures, experimental in vivo models, and in some instances, clinical trials. New techniques including slice culturing of hippocampal tissue from transgenic mice as well as more mature brain tissue, and slice cultures coupled to microelectrode arrays (MEAs), on-line biosensor monitoring, and time-lapse fluorescence microscopy are also presented.

Group I metabotropic glutamate receptors reduce excitotoxic injury and may facilitate neurogenesis.

Group I metabotropic glutamate receptor (mGluR) agonist DHPG reduced nerve cell death caused by their exposure to NMDA ("neuroprotective effect") and attenuated NMDA receptor-mediated currents [Blaabjerg, M., Baskys, A., Zimmer, J., Vawter, M. P., 2003b. Changes in hippocampal gene expression after neuroprotective activation of group I metabotropic glutamate receptors. Brain Research, Molecular Brain Research 117, 196-205.]. In the present study, we used organotypic hippocampal culture preparation to examine specific phospholipase C (PLC) inhibitor U73122 effects on DHPG-induced neuroprotection, changes in excitatory synaptic transmission associated with the neuroprotective DHPG treatment and a role of group I mGluR ligands in neurogenesis. Results show that short (10 min) DHPG treatment did not result in neuroprotection but significantly depressed field synaptic potentials (fEPSP) in the Schaffer collateral-CA1 pathway. The fEPSP depression was not affected by the PLC inhibitor U73122. In contrast, prolonged (2-h) treatment of cultures with DHPG induced a significant protective effect that was blocked by a PLC inhibitor U73122 but not by its inactive analog U73343. Voltage-clamp measurements of spontaneous miniature excitatory post-synaptic currents (EPSCs) recorded in CA1 neurons from cultures treated with DHPG (10 microM, 2 h) showed a significant reduction of the EPSC amplitude in DHPG-treated but not control (untreated) cultures. This reduction was completely abolished by U73122, suggesting a PLC involvement. Since activation of PLC is thought to be associated with cell proliferation, we investigated whether group I mGluR agonist DHPG or subtype antagonists LY367385 and MPEP have an effect on dentate granule cells expressing immature neuronal marker TOAD-64. DHPG (100 microM, 72 h) slightly but not significantly increased the number of TOAD-64 positive cells. The mGluR1 antagonists LY367385 (10 microM, 72 h) markedly decreased the number of TOAD-64 positive cells and mGluR5 antagonist MPEP (1 microM, 72 h) had no effect. These data suggest that (1) prolonged activation of group I mGluRs reduces nerve cell susceptibility to excitotoxic injury in a PLC-dependent manner; (2) this reduction is associated with a PLC-dependent depression of excitatory synaptic transmission; and (3) mGluR1 activation may facilitate neurogenesis.

Neural transdifferentiation of mesenchymal stem cells--a critical review.

The classic concept of stem cell differentiation can be illustrated as driving into a series of one-way streets, where a given stem cell through generations of daughter cells becomes correspondingly restricted and committed towards a definitive lineage with fully differentiated cells as end points. According to this concept, tissue-derived adult stem cells can only give rise to cells and cell lineages found in the natural, specified tissue of residence. During the last few years it has, however, been reported that under certain experimental conditions adult stem cells may lose their tissue or germ layer-specific phenotypes and become reprogrammed to transdifferentiate into cells of other germ layers and tissues. The transdifferentiation process is referred to as "stem cell plasticity". Mesenchymal stem cells, present in various tissues, including bone marrow, have--besides differentiation into bone, cartilage, smooth muscle and skeletal muscle--also been reported to transdifferentiate into skin, liver and brain cells (neurons and glia). Conversely, neural stem cells have been reported to give rise to blood cells. The actual occurrence of transdifferentiation is currently much debated, but would have immense clinical potential in cell replacement therapy and regenerative medicine. Controlled neural differentiation of human mesenchymal stem cells might thus become an important source of cells for cell therapy of neurodegenerative diseases, since autologous adult mesenchymal stem cells are more easily harvested and effectively expanded than corresponding neural stem cells. This article provides a critical review of the reports of neural transdifferentiation of mesenchymal stem cells, and proposes a set of criteria to be fulfilled for validation of transdifferentiation.

Activation of neuropeptide Y receptors is neuroprotective against excitotoxicity in organotypic hippocampal slice cultures.

Glutamate and NPY have been implicated in hippocampal neuropathology in temporal lobe epilepsy. Thus, we investigated the involvement of NPY receptors in mediating neuroprotection against excitotoxic insults in organotypic cultures of rat hippocampal slices. Exposure of hippocampal slice cultures to 2 microM AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate) induced neuronal degeneration, monitored by propidium iodide uptake, of granule cells and CA1 pyramidal cells. For dentate granule cells, selective activation of Y1, Y2, or Y5 receptors with 1 microM [Leu31,Pro34]NPY, 300 nM NPY13-36 or 1 microM 500 nM NPY(19-23)-(Gly1,Ser3,Gln4,Thr6,Ala31,Aib32,Gln34)-PP, respectively, had a neuroprotective effect against AMPA, whereas only the activation of Y2 receptors was effective for CA1 pyramidal cells. When the slice cultures were exposed to 6 microM kainate, the CA3 pyramidal cells displayed significant degeneration, and in this case the activation of Y1, Y2, and Y5 receptors was neuroprotective. For the kainic acid-induced degeneration of CA1 pyramidal cells, it was again found that only the Y2 receptor activation was effective. Based on the present findings, it was concluded that Y1, Y2, and Y5 receptors effectively can modify glutamate receptor-mediated neurodegeneration in the hippocampus.

Glutamate receptor antagonists and growth factors modulate dentate granule cell neurogenesis in organotypic, rat hippocampal slice cultures.

Generation of dentate granule cells and its modulation by glutamate receptor antagonists, growth factors and pilocarpine-induced seizure-like activity was investigated in rat hippocampal slice cultures derived from 1-week-old rats and grown for 2 weeks. Focussing on the dentate granule cell layer facing CA1 and the immediate subgranular zone, exposure for 3 days to the NMDA receptor blocking agents MK-801 (10 microM) or APV (25 microM) in the culture medium, increased the number of TOAD-64/Ulip/CRMP-4 (TUC-4)-positive cells as counted in the slice cultures at the end of the 3-day treatment period. Exposure to IGF-I (200 ng/ml) and EGF (20 ng/ml) also increased the number of TUC-4-positive cells. Combining APV with IGF-I/EGF had an additive effect. Similar results were obtained by 3 days treatment with the AMPA receptor antagonist CNQX (25 microM). Surprisingly, addition of 5 mM pilocarpine reduced the number of TUC-4-positive cells, just as combining pilocarpine with the neurogenesis-stimulating compounds, prevented or reduced the increase of TUC-4-positive cells. None of the treatments were found to induce dentate granule cell death within the observed period. Labeling of dividing cells by adding 5-bromo-2-deoxyuridine (BrdU) to the culture medium did not result in cells double-labeled with BrdU and TUC-4. The induced increase in TUC-4-positive cells therefore represent neuronal differentiation of existing neural precursor cells when investigated at the 3-day time point. We conclude that 3 days treatment of 2-week-old hippocampal slice cultures with IGF-I and EGF and NMDA and AMPA glutamate receptor antagonists increase granule cell neurogenesis from preexisting neural precursors.

Changes in extracellular action potential detect kainic acid and trimethyltin toxicity in hippocampal slice preparations earlier than do MAP2 density measurements.

In vitro electrophysiological techniques for the assessment of neurotoxicity could have several advantages over other methods in current use, including the ability to detect damage at a very early stage, and could further assist in replacing animal experimentation in vivo. We investigated how an electrophysiological parameter, the extracellularly-recorded compound action potential ("population spike", PS) could be used as a marker of in vitro neurotoxicity in the case of two well-known toxic compounds, kainic acid (KA) and trimethyltin (TMT). We compared the use of this electrophysiological endpoint with changes in immunoreactivity for microtubule-associated protein 2 (MAP2), a standard histological test for neurotoxicity. We found that both toxic compounds reliably caused disappearance of the PS, and that such disappearance occurred after only 1 hour of exposure to the drug. By contrast, densitometric measurements of MAP2 immunoreactivity were unaffected by both KA and TMT after such a short exposure time. We conclude that, in the case of KA and TMT, the extracellular PS was abolished at a very early time-point, when MAP2 immunoreactivity levels were still comparable to those of the untreated controls. Electrophysiology could be a reliable and early indicator of neurotoxicity, which could improve our ability to test for neurotoxicity in vitro, thus further replacing the need for in vivo experimentation.

Changes in hippocampal gene expression after neuroprotective activation of group I metabotropic glutamate receptors.

Stimulation of group I metabotropic glutamate receptors (mGluRs) has been shown to protect against N-methyl-D-aspartate receptor-mediated cell death, but the underlying cellular mechanism is unknown. Using cDNA microarrays we have now compared gene expressions in organotypic hippocampal slice cultures after neuroprotective activation of group I mGluRs with (S)-3,5-dihydroxyphenylglycine (DHPG; 10 microM, 2 h) with untreated control cultures. Total RNA was extracted from the cultures immediately after the neuroprotective treatment, reverse transcribed to cDNA with incorporation of [32]P-dCTP, and then hybridized to the arrays. Of a total of 1128 genes on the Neuroarray, 33 genes displayed significant changes in expression after DHPG-treatment (six up- and 27 downregulated). These genes have been associated with regulation of synaptic excitation, inflammation, cell adhesion, cell death, and transcription. The small GTPase RAB5B associated with endocytosis emerged as a primary candidate gene for neuroprotection, and its expression was confirmed by Western blot analysis and real time polymerase chain reaction. By providing insight into genes involved in neuroprotection these data may help to identify novel therapeutic targets.