Dentate granule neuron apoptosis and glia activation in murine hippocampus induced by trimethyltin exposure.

We investigated the effect of trimethyltin (TMT), a well-known neurotoxicant, on murine hippocampal neurons and glial cells. Three days following intraperitoneal (i.p.) injection of TMT into 1-month-old Balb/c mice at a dose of 2.5 mg/kg body weight we detected damage of the dentate gyrus granular neurons. The dying cells displayed chromatin condensation and internucleosomal DNA fragmentation, which are the most characteristic features of apoptosis. To study, if prolyl oligopeptidase is engaged in neuronal apoptosis following TMT administration, we pretreated mice with the specific inhibitor--Fmoc-Pro-ProCN in doses of 5 and 10 mg/kg body weight (i.p. injection). Three days following injection we did not observe any attenuation of neurotoxic damage, regardless of inhibitor dose, indicating the lack of prolyl oligopeptidase contribution to neuronal injury caused by TMT. The neurodegeneration was associated with reactive astrogliosis in whole hippocampus, but particularly in injured dentate gyrus. The reactive astrocytes showed an increased nerve growth factor (NGF) expression in ventral as well as dorsal hippocampal parts. NGF immunoreactivity was also augmented in neurons of CA3/CA4 areas, which were almost totally spared after TMT intoxication. It suggested a role for this neurotrophin in protection of pyramidal cells from loss of connection between CA3/CA4 and dentate gyrus fields. The granule neurons' death was accompanied by increased histochemical staining with isolectin B4, a marker of microglia, in the region of neurodegeneration. The microglial cells displayed ramified and ameboid morphology, characteristic of their reactive forms. Activated microglia were the main source of interleukin 1beta (IL-1beta). It is possible that this cytokine may participate in neurodegeneration of granule cells. Alternatively, IL-1beta elaborated by microglia could play a role in increasing NGF expression, both in astroglia and in CA3/CA4 neurons.

Treatment of hippocampal neurons with cyclosporin A results in calcium overload and apoptosis which are independent on NMDA receptor activation.

Calcineurin is a ubiquitous calcium/calmodulin dependent protein phosphatase that has been shown to regulate the activity of ion channels, glutamate release, and synaptic plasticity. In the present study we show that CsA, a specific inhibitor of calcineurin, affects the survival of cultures developed from hippocampal dentate gyrus. Mixed neuronal-glial cultures exposed to 8 - 40 microM CsA undergo cell death characterized by apoptotic changes in cellular and nuclear morphology. TUNEL-positive staining was observed only in neurons that developed pyknotic morphology after treatment with 8 microM CsA for 24 - 72 h. Immunocytochemical staining with an anti-GFAP monoclonal antibody revealed that astrocytes from mixed neuronal/glial cultures were unaffected by exposure to CsA at doses toxic for neurons and all TUNEL-positive cells were neurons. MK-801, a noncompetitive inhibitor of glutamate receptor, does not inhibit the appearance of TUNEL-positive neurons and apoptotic changes in nuclear morphology. Preincubation of cells with 8 microM CsA increased basal intracellular calcium level in time dependent manner and decreased relative calcium response to glutamate. Application of 1 microM MK-801 had no effect on CsA-induced changes in Ca(2+) level. Our findings suggest that the neuronal death after CsA treatment is not a result of glutamate excitotoxicity and the increase in intracellular calcium concentration in neurons is not dependent on calcium influx via NMDA channel.

Efficient expression of tetracycline-responsive gene after transfection of dentate gyrus neurons in vitro.

Gene transfer into neurons both in vivo and in vitro may aid in understanding of gene regulation and function in nerve cells. Especially desirable is ability to control the gene expression. In this study we developed conditions for transfection of hippocampal dentate gyrus neurons in dissociated cultures in vitro by calcium-phosphate method. Furthermore, we describe an effective use of tetracycline responsive gene promoter (Tet-On) system for the controlled and very efficient expression of transfected genes. Under optimal conditions as established in this study, efficiency of transfection of neurons with green fluorescent protein (GFP) driven by constitutive cytomegalovirus (CMV) early promoter reached 2.7%. With tetracycline responsive promoter percentage of GFP-positive neurons raised in the presence of tetracycline analog, doxycycline up to 20%. Application of the Tet-On system resulted in almost 10-fold induction of GFP expression.

Neuronal excitation-driven and AP-1-dependent activation of tissue inhibitor of metalloproteinases-1 gene expression in rodent hippocampus.

Understanding of biological function of AP-1 transcription factor in central nervous system may greatly benefit from identifying its target genes. In this study, we present several lines of evidence implying AP-1 in regulating expression of tissue inhibitor of metalloproteinases-1 (timp-1) gene in rodent hippocampus in response to increased neuronal excitation. Such a notion is supported by the findings that timp-1 mRNA accumulation occurs in the rat hippocampus after either kainate- or pentylenetetrazole-evoked seizures with a delayed, in comparison with AP-1 components, time course, as well as with spatial overlap with c-Fos protein (major inducible AP-1 component) expression. Furthermore, AP-1 sequence derived from timp-1 promoter is specifically bound by hippocampal AP-1 proteins after treating the rats with either pro-convulsive agent. Finally, timp-1 promoter responds to excitatory activation both in vivo, in transgenic mice harboring the timp-LacZ gene construct, and in vitro in neurons of the hippocampal dentate gyrus cultures. These findings suggest that the AP-1 transcription factor may exert its role in the brain through affecting extracellular matrix remodeling.

Cellular and molecular correlates of glutamate-evoked neuronal programmed cell death in the in vitro cultures of rat hippocampal dentate gyrus.

An excessive neuronal stimulation through glutamate receptors is known to result in excitotoxic cell death of apoptotic (programmed) character. Granule cells of hippocampal dentate gyrus are believed to be particularly resistant to excitotoxic insults, despite the fact that pyramidal neurons of the hippocampus proper are apparently the most vulnerable brain cells. In this study, we report that neurons derived from the rat 5-day-old dentate gyrus, and maintained in vitro for 6 days, may undergo apoptosis after treatment with L-glutamate, in a dose-dependent manner-with up to 80% of neurons displaying features of programmed cell death after 24 h exposure to 0.5 mM glutamate. This conclusion is based on morphological evaluation of the cultures, nuclear staining with Hoechst 33258 and acridine orange revealing chromatin abnormalities, as well as terminal transferase labeling of DNA fragmentation. Since apoptosis is believed to be an active process involving gene expression, immunocytochemical of c-Fos and c-Jun transcription factor proteins was performed. Elevated expression of both proteins was found to follow quickly (within 1 h) after addition of glutamate. However, this effect was not dose-dependent, thus it does not provide clear correlations to the programmed cell death. In conclusion, this study reports on the establishment of a novel apoptotic model of excitotoxicity, and invites further efforts to investigate a basis for in vitro susceptibility and in vivo resistance of dentate gyrus granule cells to excitotoxic insult evoking apoptosis.

Orthovanadate induces cell death in rat dentate gyrus primary culture.

The aim of this study was to define the effects of a potent inhibitor of tyrosine phosphatases, sodium orthovanadate (0.1-100 microM for up to 48 h), on dentate gyrus cells (DGC) in culture. Treatment with 100 microM orthovanadate evoked a delayed form of cell death. To examine the possible involvement of apoptosis in orthovanadate-induced cell death, biochemical and morphological alterations were compared with those of necrotic death induced by sodium azide. Phase-contrast microscopy and nuclear condensation analysis showed that orthovanadate and azide each evoked cell death by distinct pathways. TUNEL assay was positive in both cases. Application of a protein synthesis inhibitor, cycloheximide, did not prevent cytotoxicity caused by either orthovanadate or azide and potentiated the effects of vanadate. We conclude that orthovanadate-induced death of DGC bears features of apoptosis.

Cyclosporin A, an immunosuppressive drug, induces programmed cell death in rat C6 glioma cells by a mechanism that involves the AP-1 transcription factor.

Cyclosporin A (CsA) is a clinically important immunosuppressive drug widely used to prevent graft rejection following organ or bone marrow transplantation. Although there are reports of serious neurologic alterations associated with the use of the drug, the precise mechanism of its action on the CNS still remains unknown. We studied the effects of CsA on the growth of C6 glioma cells. We found that CsA inhibits the growth of C6 glioma cells in a dose-dependent manner and induces morphological changes such as shrinkage of the cell body and loss of extensions followed by cell death. The analysis of DNA from CsA-treated cells revealed a ladder-like pattern of fragmented DNA. Acridine orange staining showed the occurrence of apoptotic changes in nuclear morphology. Apoptotic morphological alterations were prevented by the treatment with cycloheximide. Altogether, our findings suggest that the CsA-induced cell death of C6 glioma cells bears all the features characteristic of programmed cell death. We also observed a significant increase in the DNA-binding activity of AP-1 during CsA-induced apoptosis. The AP-1 induction preceded the appearance of apoptotic, morphological changes and was accounted for by an increase in the expression of c-Jun protein. The occurrence of increased levels of AP-1 complex and c-Jun protein during CsA-induced programmed cell death suggests its involvement in the induction of apoptosis.

A new model for the research into rhythmic contraction activity of cardiomyocytes in vitro.

Heart cells continue to contract rhythmically after isolation and in culture in vitro. We describe a model of heart preparation in vitro that permits quantitative research on the frequency of contractions of cardiomyocytes. The chick embryo heart explants placed on a network of elastic glass fibers continued beating for months, recorded and analyzed with the methods of computer-assisted image analysis. The efficacy of this experimental model for the screening of effects of various agents on the frequency of contractions was examined by following the effects of nifedipine, caffeine, ethanol, and benzamide. The reversibility of the effects and the reproducibility of results were demonstrated quantitatively. The significance of a mechanical elastic load provided by glass fibers for the preservation of long-lasting contractile activity of cardiomyocytes is discussed and the common occurrence of oscillatory contraction processes in various eucaryotic cells is noted.

Studies on effects of culture conditions and age of donor on hippocampal neurons in vitro.

The hippocampus is a brain structure of pivotal role in many physiological and pathological responses including memory formation, epilepsy and ischemia. Understanding of these processes requires a good knowledge of hippocampal neurons. In vitro culture offers a tool to study these cells in a controlled environment. In order to optimize culture conditions several growth parameters were investigated in hippocampal neuronal cultures derived from rats of different age: 18-day old fetuses, newborns and 5-day old rat pups, maintained in either serum-containing or chemically defined media. Usefulness of particular culture conditions for special purposes is discussed.