Izabela Figiel

New hippocampal neurons are not obligatory for memory formation; cyclin D2 knockout mice with no adult brain neurogenesis show learning

The role of adult brain neurogenesis (generating new neurons) in learning and memory appears to be quite firmly established in spite of some criticism and lack of understanding of what the new neurons serve the brain for. Also, the few experiments showing that blocking adult neurogenesis causes learning deficits used irradiation and various drugs known for their side effects and the results obtained vary greatly. We used a novel approach, cyclin D2 knockout mice (D2 KO mice), specifically lacking adult brain neurogenesis to verify its importance in learning and memory. D2 KO mice and their wild-type siblings were tested in several behavioral paradigms, including those in which the role of adult neurogenesis has been postulated. D2 KO mice showed no impairment in sensorimotor tests, with only sensory impairment in an olfaction-dependent task. However, D2 KO mice showed proper procedural learning as well as learning in context (including remote memory), cue, and trace fear conditioning, Morris water maze, novel object recognition test, and in a multifunctional behavioral system-IntelliCages. D2 KO mice also demonstrated correct reversal learning. Our results suggest that adult brain neurogenesis is not obligatory in learning, including the kinds of learning where the role of adult neurogenesis has previously been strongly suggested.

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.

Cyclosporin A, an Immunosuppressive Drug, Induces Programmed Cell Death in Rat C6 Glioma Cells by a Mechanism that Involves the AP‐1 Transcription Factor

Abstract: 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.

TNFα and TNF receptor 1 expression in the mixed neuronal-glial cultures of hippocampal dentate gyrus exposed to glutamate or trimethyltin

We examined the expression and cellular localization of tumor necrosis factor alpha (TNFalpha) and its type 1 receptor (TNFR1) in mixed neuronal-glial cultures of rat hippocampal dentate gyrus exposed to glutamate (GLU) or trimethyltin (TMT). Our previous studies demonstrated that both pathogenic factors evoked neuronal apoptosis, however, TMT was more potent and caused cell death in almost 90% of neurons. Observed neurodegeneration was accompanied by morphological changes of microglia. In the current study, using RT-PCR and Western blotting analysis, we found that GLU and TMT induced increase in TNFalpha mRNA and protein levels. The induction of transcription was stronger following GLU treatment, however the protein production was much more intensive after TMT exposure. Double fluorescent labeling for TNFalpha, TNFR1 and cellular markers revealed cytokine expression in microglia and some neurons. On the other hand, majority of neuronal cells displayed TNFR1 immunoreactivity, in control and in treated cultures. Moreover, TMT led to a strong increase in TNFR1 expression in astrocytes, which displayed remarkable, granular staining for the cytokine receptor. Western blotting for TNFR1 revealed enhanced protein expression only in cultures treated with TMT. This is the first report demonstrating the changes of expression of TNFalpha and TNFR1 in hippocampal dentate gyrus cultures treated with GLU or TMT. Our results indicate that TNFalpha may be involved in the mechanism of neurotoxic effects evoked by both pathogenic factors and suggest that astrocytes via TNFR1 may enhance TMT-induced injury.

Transient brain ischemia due to cardiac arrest causes irreversible long-lasting cognitive injury.

Herein, we used a clinically-relevant model of 10 min cardiac arrest (CA) in Wistar rats. Histological analyses of the ischemic brains of old rats showed significant atrophy of CA(1) sector of hippocampus (Nissl and NeuN stainings) corresponding with increase of glial fibrillary acidic protein expression. The long-term behavioral consequences of above manipulation producing global brain ischemia were assessed in young, middle-aged and old rats, i.e., 3-, 6- and 18-months post-treatment, respectively. In young animals no differences were found in the context-dependent memory in Fear Conditioning test. The most striking behavioral abnormalities were found in middle-aged rats (6 months post-ischemia). Ischemic rats showed hyperactivity and decreased level of anxiety in Open Field and problems with spatial learning and memory in a Novel Object Location test, T-maze and Morris Water Maze. In old animals, a decline of motor and cognitive functions was found not only in ischemic but also in sham/control ones. This study describes consequences of global brain ischemia in aging animals.

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.

The ameboid phenotype of NG2 (+) cells in the region of apoptotic dentate granule neurons in trimethyltin intoxicated mice shares antigen properties with microglia/macrophages

NG2+, stellate cells present in the adult central nervous system (CNS) have been recently recognized as a distinct glial class, identified as multipotent progenitor cells. Antigenically, they are indistinguishable from oligodendroglia progenitor cells. In response to a variety of CNS insults, these cells become rapidly activated and undergo morphological changes accompanied by increased cellular proliferation. The role they play with respect to injured neurons is not clear. In our studies, we performed immunocytochemical investigations and identified a response of NG2‐expressing cells in the model of selective neurodegeneration of murine dentate gyrus granule cells induced by systemic administration of trimethyltin. Dying neurons exhibited features of apoptotic cells. Around the region of neurodegeneration, we observed activation of NG2+ stellate cells and microglia. During the peak of apoptosis, we detected the appearance of NG2+ cells of the ameboid phenotype, intermingled with granule neurons. These cells also expressed markers of microglia/macrophages, OX42‐ and ED1‐recognized antigens, an antigen recognized by O4 antibody—a marker of more differentiated cells of the oligodendroglia lineage and, in some cases, also a protein of mature oligodendroglia adenomatus polyposis coli. They also expressed nestin. Our results suggest that the injury induces a parallel transformation of both the activated glial classes: NG2+ stellate cells and resident microglia, into ameboid cells, sharing properties of both oligodendrocyte and monocyte lineages. These cells may play a role in the phagocytosis. If this assumption is verified by electron microscopy, it would indicate a novel function of NG2 transformed cells under CNS injury conditions.

Trimethyltin-evoked neuronal apoptosis and glia response in mixed cultures of rat hippocampal dentate gyrus: a new model for the study of the cell type-specific influence of neurotoxins.

We investigated the effects of a potent neurotoxin, trimethyltin (TMT), on mixed neuronal/glial cultures derived from rat hippocampal dentate gyrus. We found that TMT induced neuronal cell death in a concentration dependent manner, which was estimated by microtubule degeneration, hematoxylin histological staining and the TUNEL method. This cell death is most probably of an apoptotic type as suggested by Hoechst staining. In parallel to studies the effects of TMT on neurons, its concentration dependent actions on astroglia and microglia were also examined using GFAP and GS-B4 isolectin as immunocytochemical markers, respectively. We found that neurotoxic concentrations of TMT evoked astrocytic swelling, whereas low, non-cytotoxic concentrations caused changes in microglia morphology characteristic of their active form. The combined results of our studies provide new data concerning the cell type-specific influence of TMT and indicate that the culture of dentate gyrus cells is a feasible in vitro modelforfurther studies of neuronal-glial interaction in response to toxic injury.

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 μM 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 μM 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 μM CsA increased basal intracellular calcium level in time dependent manner and decreased relative calcium response to glutamate. Application of 1 μM MK‐801 had no effect on CsA‐induced changes in Ca2+ 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.

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 orthovana-date (0.1–100 μM for up to 48 h), on dentate gyrus cells (DGC) in culture. Treatment with 100 μM orthovana-date 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 orthovana-date 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 cytotoxicicity caused by either orthovanadate or azide and potentiated the effects of vanadate. We conclude that orthovanadate-induced death of DGC bears features of apoptosis.